"    '    '•- 


GIFT  OF 
Professor  W*A»Setchell 


BIOLOGY  LIBRARY 


W1LUAM  A.  SETCHELL 


STATE  OF  ILLINOIS 

DEPARTMENT  OF  REGISTRATION  AND  EDUCATION 
DIVISION  OF  THE 

NATURAL  HISTORY  SURVEY 

STEPHEN  A.  FORBES,  Chief 

Vol.  XIV.  BULLETIN  Article  V. 

The  Helminthosporium  Foot-rot  of  Wheat,  with 

Observations   on   the   Morphology  of 

Helminthosporium   and  on  the 

Occurrence  of  Saltation 

in  the  Genus 

BY 
F.  L.  STEVENS 


PRINTED  BY  AUTHORITY  OF  THE  STATE  OF  ILLINOIS 


URBANA,  ILLINOIS 
June,  1922 


Original  isolations  of  the  foot-rot  Helminthosporium  made 

in  May,   1919,  from  bits  of  tissue  from  wheat 

grown  in  Madison  county,  Illinois. 


STATE  OF  ILLINOIS 
DEPARTMENT  OF  REGISTRATION  AND  EDUCATION 

DIVISION  OF 

NATURAL  HISTORY  SURVEY 

STEPHEN  A.  FORBES,  Cheif 

Vol.  XIV.  BULLETIN  Article  V, 

The  Helminthosporium  Foot-rot  of  Wheat,  with 

Observations    on    the    Morphology   of 

Helminthosporium  and  on  the 

Occurrence  of  Saltation 

in  the  Genus 

BY 
F.  L.  STEVENS 


PRINTED  BY  AUTHORITY  OF  THE  STATE  OF  ILLINOIS 


URBANA,  ILLINOIS 
June,  1922 


LIBRA*! 

a 


GIFT  OF 


BIOLuuY 


STATE  OF  ILLINOIS 

DEPARTMENT  OF  REGISTRATION  AND  EDUCATION 
W.  H.  H.  MILLER,  Director 


BOARD  OF 

NATURAL  RESOURCES  AND  CONSERVATION 
W.  H.  H.  MILLER,  Chairman 


WILLIAM  TRELEASE,  Biology 
JOHN  M.  COULTER,  Forestry 
ROLLIN  D.  SALISBURY,  Geology 
WILLIAM  A.  NOYES,  Chemistry 


JOHN  W.  ALVORD,  Engineering 

KENDRIC  C.  BABCOCK,  Representing  the 
President  of  the  University  of  Illi- 
nois 


THE  NATURAL  HISTORY  SURVEY  DIVISION 
STEPHEN  A.  FORBES,  Chief 


(52929-1200-7-21) 


CONTENTS 

PAGE 

Introductory 77 

I.    A  foot-rot  of  wheat  and  its  causal  fungus: 

Symptoms 78 

Fungi  present 78 

Growth  of  the  causal  fungus  on  various  media 79 

Various  agars  as  media 80 

Summary  concerning  growth  on  agars 85 

Rice  and  similar  substances  as  media,  with  special  note  of  color  phenomena  86 

Summary  concerning  growth  on  rice  and  similar  substances 88 

Miscellaneous  vegetable  media 88 

Summary  concerning  the  foregoing  vegetable  media 90 

Cereal  shoots  grown  from  aseptic  seeds  as  media 90 

Environmental  factors  which  induce  variation 91 

Quantity  of  nutriment 91 

Inhibitory  influences .      .  93 

Humidity  of  media 93 

Humidity  of  air                             > 94 

Temperature  relations 98 

Light 99 

Carbohydrates 100 

Nutrients  as  affecting  conidial  length,  septation,  and  shape 101 

Summary  concerning  environmental  factors  which  induce  variation    .      .      .  102 

Morphology  of  the  foot-rot  fungus 103 

Mycelium 105 

Senescence  phenomena  of  aerial  mycelium 107 

Conidiophores 109 

Conidia 110 

Etiology  of  foot-rot: 

Evidences  of  etiological  relation  of  H.  No.  1         124 

Constant  presence  of  the  pathogene 124 

Absence  of  other  constant  parasites 124 

Identity  of  pathogene  proved  by  culture 124 

Evidence  of  infectiousness 124 

Conidia  produced  in  moist-chamber  culture 125 

Evidence  from  inoculation 125 

Recovery  of  organism 128 

Infection  phenomena  on  wheat 128 

Susceptibility  of  various  hosts  to  infection 137 

Summary  concerning  etiology  of  foot-rot 139 


IV 

II.     Evidence  and  discussion  of  the  occurrence  of  saltation  within  the  genus  Helmin- 

thosporium :  PAGE 

Introductory 139 

Characters  of  saltants  as  shown  in  transfers 141 

Tendencies  in  saltation 145 

Stability  of  the  saltants 145 

Stability  of  the  saltants  through  the  conidia 146 

Apparent  reversions 147 

Supposititious  causes  of  the  variant  sectors 147 

Saltations  from  single  conidia 149 

Frequency  of  saltation        150 

Saltations  occurred  on  various  media 152 

Saltations  and  modifications  occurring  in  test-tube  cultures    .      .      .      .      .      .  152 

Saltations  in  nature .  154 

Notes  concerning  selected  individual  saltants 155 

General  discussion  of  saltation 157 

Taxonomy 164 

Conclusion                             ^ 168 

Summary 168 

Literature  cited 171 

Appendix: 

Methods 179 

List  of  Helminthosporiums  used  for  purposes  of  comparison 181 

Discussion  of  foregoing  list  with  several  brief  descriptions 184 

Graphs:     Figures  A — Y. 

Plates:     VII— XXXIV. 


ARTICLE  V. — The  Helminthosporium  Foot-rot  of  Wheat,  with  Observations 
on  the  Morphology  of  Helminthosporium  and  on  the  Occurrence  of  Saltation  in 
the  Genus.  By  F.  L.  STEVENS. 


INTRODUCTCXRIT  /    ,         -,^ 

The  present  study  of  wheat  disease  is^baseti  upon  Vfo6t-fot/6r  rot  of 
the  basal  portion  of  the  stems,  of  wheat  plants,  as  it  occurred  in  Madison 
county,  Illinois,  in  1919  and  subsequently.  This  disease  was  first  reported 
in  United  States  Government  publications  as  " take-all"  ( Ophiobolus  gram- 
inis] ;  later,  merely  as  "take-all,"  no  cause  being  assigned;  and  for  some  time 
past,  in  Government  publications  it  has  usually  been  designated  as  "so- 
called  take-all."  An  annotated  bibliography  of  nearly  one  hundred 
titles  concerning  foot-rot  disease  of  wheat,  prepared  by  the  writer,  was 
presented  before  the  Cereal  Pathologists  of  America  at  St.  Louis  in  June, 
1919,  and  this,  expanded  to  one  hundred  and  eighty-eight  titles,  was 
published  in  October,  1919  (116).  As  early  as  May,  1919,  cultural  studies 
quite  clearly  pointed  to  Helminthosporium  as  the  true  cause  of  the  disease, 
and  at  the  December  meeting  of  the  American  Phytopathological  Society 
I  announced  this  fungus  as  the  probable  cause.  In  May,  1920,  in  a  note  in 
Science  (117),  I  published  the  statement  that  it  had  been  conclusively 
established  that  this  foot-rot  of  wheat  is  caused  by  Helminthosporium. 
One  purpose  of  the  present  paper  is  to  present  the  evidence  on  which 
the  foregoing  conclusion  is  based  and  certain  facts  concerning  the  mor- 
phology and  parasitism  of  the  fungus;  but  far  transcending  in  interest  the 
disease  itself — which  now  appears  to  be  one  of  much  less  alarming  nature 
than  was  at  first  feared — is  the  fact  that  very  striking  phenomena  of 
variability  are  found  in  this  and  related  fungi.  In  the  following  pages, 
therefore,  appear  (I)  an  account  of  the  Illinois  foot-rot  of  wheat  and  its 
causal  fungus;  and  (II)  evidence  and  discussion  of  the  occurrence*of  salta- 
tion within  the  genus  Helminthosporium. 

ACKNOWLEDGMENTS 

In  this  study  I  have  been  assisted  financially  by  grants  from  the 
Illinois  Natural  History  Survey  and  from  the  University  of  Illinois.  I 
am  indebted  for  specimens  to  persons  mentioned  in  the  list  of  species 
used  for  comparison  (pages  181-184),  and  to  W.  P.  Snyder  for  compu- 
tation of  data  embodied  in  several  of  the  graphs.  I  wish  also  to  express 
my  thanks  to  Prof.  J.  A.  Detlefsen,  who  kindly  read  the  manuscript  and 
offered  valuable  suggestions  regarding  genetic  questions. 


78 
I.     A  Foot-rot  of  Wheat  and  its  Causal  Fungus 

SYMPTOMS 

As  the  name  implies,  the  most  obvious  symptom  is  a  rotting  of  the 
basal  portion  of  the  stem  of  the  wheat  plant,  that  is,  the  lowesr  portion 
of  the  lowest  internode.  In  earlier  stages  than  that  of  actual  rotting, 
minute  yellow  or  brown  lesions  occur  on  the  stem  (PI.  VII),  while 
the  roots,  if  diseased,  are  slightly  yellowed  and  largely  or  quite  devoid 
of  functional  root-hairs.  No  weft  of  superficial  mycelium  or  black  incrus- 
tation, such  as  is  so  frequently  described  in  articles  concerning  take- 
all,  was  seen.  The  diseased  tissues,  however,  were  invariably  ramified 
by  an  internal  mycelium.  Certain  cases  of  diseased  wheat  came  under 
observation  in  which  the  plants  had  attained  a  nearly  normal  growth 
and  were  eighteen  inches  high,  when  they  suddenly  died  throughout. 
In  such  cases  there  was  a  slight  darkening  of  the  lower  node  and  a  mycelial 
invasion  at  this  point.  The  opinion  of  those  who  observed  this  wheat 
in  the  field  was  that  the  death  was  due  to  frost  injury.  It  is  probable 
that  the  actual  cause  of  death  was  foot-rot  following  the  frost  injury. 

FUNGI  PRESENT 

Direct  microscopic  observation  of  the  diseased  tissues,  in  all  cases 
of  foot-rot  examined,  revealed  the  presence  of  an  internal  hyaline  or 
faintly  tinted  mycelium  in  great  abundance  permeating  the  diseased 
tissues.  Mycelium  of  different  character  was  also  occasionally  found, 
but  so  inconstantly  as  apparently  to  have  no  actual  relation  to  the  disease. 
Isolations  of  the  fungi  present  in  the  diseased  tissues  were  made  by  two 
methods: 

1.  Direct  planting  of  bits  of  diseased  tissue  on  poured  agar  (corn- 
meal  agar  or  wheat-straw  agar).     The  diseased  tissue  was  secured  in  as 
clean  condition  as  possible  by  stripping  back  the  enclosing  sheaths,  ex- 
cising the  diseased  part  with  sterile  tools,  and  tearing  it  apart  in  a  sterile 
Petri-dish. 

2.  Direct  planting  of  similar  bits  of  diseased   tissue  after  surface- 
sterilization  in  mercuric  chloride  (1-1000,  10  min.). 

Dilution  plating  was  unsatisfactory  owing  to  the  paucity  of  conidia 
and  the  presence  of  numerous  soil  bacteria,  particularly  "spreaders." 

As  might  be  expected,  the  methods  employed  gave  rise  to  colonies 
of  many  genera  and  species,  including  Phyllosticta,  Septoria,  Fusarium, 
Epicoccum,  Alternaria,  and  Helminthosporium.  A  striking  fact,  however, 


79 

was  that  with  the  exception  of  the  Helminthosporium,  these  fungi  were 
very  rarely  present,  and  then  only  a  single  colony  or  part  of  a  mixed  colony 
on  occasional  plates.  Alternaria  occurred  with  remarkable  rarity;  only 
two  or  three  colonies  among  several  thousand.  Fusarium  was  found  in 
only  a  few  colonies  and  so  mixed  that  it  was  isolated  with  difficulty.  Ep- 
icoccum  occurred  in  two  colonies;  Phyllosticta  also  in  two  colonies  (two 
species) . 

A  Helminthosporium,  however,  appeared  in  every  plate  and  from 
nearly  every  bit  of  tissue  used,  no  matter  how  great  the  care  in  securing  the 
inoculum.  On  many  plates  this  Helminthosporium  (which  throughout 
this  article  I  designate  as  H.  No.  1)  appeared  in  pure  culture  Thus  it 
may  be  said  that  the  Helminthosporium  was  universally  present  in  the 
plates;  that  it  was  the  only  organism  that  was  present  with  any  constancy; 
and  that  all  other  fungi  were  obviously  strays.*  Though  conidia  were 
never  found  in  great  numbers  on  plants  brought  in  direct  from  infested 
fields,  when  the  plants  were  placed  in  moist  chamber  for  two  or  three 
days  conidia  developed  in  abundance.  This  was  also  the  case  with  portions 
of  wheat  stems  which  had  been  placed  in  bichloride  of  mercury  for  ten 
minutes  and  then  placed  in  moist  chamber  for  several  days.  In  passing 
it  may  be  remarked  that  although  great  numbers  of  nematodes  and  amebae 
appeared  in  the  plates  there  is  no  reason  to  believe  that  they  had  any  relation 
to  the  disease  under  discussion  or  to  any  diseased  condition. 

GROWTH  OF  THE  CAUSAL  FUNGUS  ON  VARIOUS  MEDIA 

Since  the  characters  exhibited  by  various  Helminthosporiums  when 
growing  in  artificial  culture  have  been  considered  as  of  importance  as  a 
means  of  distinguishing  one  species,  variety,  race,  or  strain  from  another, 
many  media  were  employed  in  the  present  study.  This  was  done  in  part  for 
the  purpose  of  comparing  the  growth  characters  of  the  Helminthosporium 
with  characters  reported  by  others  in  connection  with  other  forms;  in  part 
with  the  hope  that  some  of  the  media  tested  might  give  emphasis  to  cer- 
tain characters  and  thus  serve  to  differentiate  between  species  or  strains 
of  the  forms  under  observation. 

The  following  notes  are,  in  the  main,  statements  of  the  characters 
presented  by  the  foot-rot  Helminthosporium  (H.  No.  1),  though  for  the 
purpose  of  comparison  notes  are  added  regarding  the  growth  of  several 


*A  letter  from  Professor  Hoffer  written  in  May,  1919,  tells  me  of  a  similar  result  from  platings  of  wheat 
foot-rot  from  Indiana,  and  similar  reports  reach  me  from  several  other  sources. 


80 

species  or  strains  of  Helminthosporium.  These  are  throughout  referred  to 
by  number  rather  than  by  name,  partly  for  brevity  and  partly  because 
the  species  of  many  of  the  races  had  not  been  determined,  while  in  some 
cases  the  names  were  of  more  or  less  doubtful  reliability.  That  the  reader 
may  formulate  his  own  judgment  of  these  forms,  introduced  for  comparison, 
a  complete  list  of  them  is  given  in  the  appendix  (pages  181-184)  together 
with  certain  notes  regarding  them. 

VARIOUS    AGARS    AS    MEDIA 

Corn-meal  agar  in  Petri  dishes. — This  medium,  prepared  after  the  di- 
rections given  by  Shear  and  Stevens  (104),  was  found  to  be  admirably 
suited  to  Helminthosporium  and  was  the  medium  chiefly  used. 

The  fungus  grew  rapidly,  the  colony  being  at  first  nearly  hyaline 
both  in  the  submerged  and  aerial  parts,  but  when  a  diameter  of  about  2-3 
cm.  was  attained  the  whole  colony  became  much  darker.  Profusion  of 
conidia  was  the  chief  factor  in  giving  the  dark  hue  to  a  colony,  the  slight 
darkening  of  the  mycelium  having  little  to  do  with  it.  The  aerial  mycelium 
varied  largely  with  change  of  conditions,  sometimes  being  very  scant 
and  at  other  times  5-6  mm.  high,  with  windrow  effects  corresponding 
with  the  zones.  After  the  colony  was  about  3  cm.  in  diameter  zonation 
became  quite  pronounced,  the  zones  corresponding  approximately  with 
the  growth  of  each  day.  At  room-temperature  the  colony  attained  a 
diameter  of  about  4.5  cm.  in  six  days.  Conidia-production  was  quite 
uniform  over  the  surface  of  the  colony  unless  checked  by  some  growth- 
inhibiting  cause,  as  drying,  cold,  or  the  antagonism  of  another  colony 
near  by,  when  it  was  much  increased,  as  evidenced  to  the  eye  by  black 
bands  in  such  regions.  By  transmitted  light  the  mycelium,  and  to  some 
extent  the  conidia  at  certain  ages,  had  a  distinctly  greenish  tinge.  H.  No.  1 
could  be  distinguished  from  H.  Nos.  5-8,  which  were  paler  and  produced 
fewer  conidia.  H.  No.  6  approached  nearer  to  H.  No.  1  in  these  regards 
than  did  the  others.  H.  Nos.  3,  4  (see  PI.  IX),  6,  15-17,  and  18  typically 
developed  more  aerial  white  mycelium  than  did  H.  Nos.  1  and  14.  H.  No. 
36  was  of  very  distinct  character  owing  to  large  development  of  aerial 
mycelium  (see  PI.  X). 

Corn-meal  agar  in  Freudenreich  flasks. — The  flasks,  of  about  100  c.c. 
capacity,  each  received  50  c.c.  of  agar  and  were  slanted.  The  large  amount 
of  nutriment  available  and  the  sustained  moisture  gave  noteworthy 
characters.  At  7  days,  with  H.  No.  1,  the  surface  of  the  slant  was  com- 


81 


pletely  overgrown  and  of  an  even  black  color,  largely  curtained  by  an 
abundant,  even  overgrowth  of  white  aerial  mycelium.  At  contact  with 
the  glass  a  sharp,  black  line  gave  clear  evidence  of  the  black  surface-coat. 
No  clumps  or  balls  of  mycelium  were  present.  At  22  days  a  few  clumps 
developed,  though  not  so  many  as  on  H.  Nos.  9,  13-16. 

Cultures  of  H.  No.  1  on  corn-meal  agar  in  large  flasks,  as  those  of 
Kolle  or  of  Piorkowski  (PL  XI-XIII)  gave  colonies  very  different  from  those 
on  the  ordinary  Petri  dish,  due  presumably  to  the  larger  quantity  of 
nutrient  available  and  to  different  humidity  relations.  These  flasks 
gave  increased  density  of  colony  and  conidia-formation,  more  aerial 
mycelium,  and  some  clumping  of  the  mycelium.  Though  colonies  of  H. 
No.  3  and  H.  No.  1  differed  in  these  characters  in  these  flasks  (PI.  XII, 
XIII),  portions  of  the  colonies  of  these  strains  were  indistinguishable. 

Corn-meal  agar  made  at  various  temperatures. — Corn-meal  agar  was 
made  in  the  usual  manner  excepting  that  the  temperature  in  three  cases 
was  held  at  43°,  85°,  and  100°  respectively,  instead  of  at  60°,  before 
filtering.  Duplicate  plates  were  made.  The  four  resulting  agars  are 
designated  according  to  the  temperatures  held,  and  colony  data  for  each 
are  presented  in  the  following  table. 

CORN-MEAL  AGAR  MADE  AT  VARIOUS  TEMPERATURES 


Temperature 

Growth  in 
8  days 

Zonation 

Density 

Colors 

43° 
43° 

7.5 
7.2 

distinct 

thin 

pale 

60° 
60° 

5.5 
6 

sharp 

thick 

dark 

85° 

6.5 

In  above  characters,  ranks  between  43°  and  60°  agars 

100° 
100° 

8 
7.8 

none 

very  thin 

very  pale 

The  100°  agar  is  most  favorable  to  linear  growth,  43°  agar  stands 
next;  43°  and  85°  agars  give  growth  of  poorer  color  than  60°  agar,  but 
100°  agar  ranks  lowest  in  this  regard.  Color  is  directly  due  to  quantity 
of  conidia,  and  it  is  uniform  in  the  mycelium  on  the  four  agars.  Nutrition 
in  100°  agar  was  very  little  better  than  in  plain  agar.  In  general,  it 


82 

appears  that  their  order  of  nutritive  value  for  this  fungus,  from  poorest 
to  best,  is  100°,  43°,  85°,  60°.  Evidently  a  temperature  of  43°  is  in- 
sufficient to  extract  the  nutrient  proteids  sufficiently,  while  100°  pre- 
cipitates too  many  of  them.  While  leucosin,  a  prominent  proteid  of  the 
embryo,  is  largely  precipitated  at  52°  and  a  second  coaguIuriTgOes  down 
at  82°,  no  more  is  precipitated  even  by  boiling  (Osborne,  89). 

Graphs  1-4  (Fig.  A),  indicating  conidial  length  on  these  four  agars, 
show  that  although  the  quantity  of  conidia  produced  varied  materially,  the 
length  and  general  variability  are  not  greatly  influenced  by  varying  the 
composition  of  the  agar — done  in  this  case  by  change  of  temperature. 
The  conidial  length  of  all  these  agars  is,  however,  considerably  less  than  that 
on  wheat  shoots  (cf.  graphs  in  Fig.  A  and  Fig.  K).  Graphs  of  conidial 
breadth  and  septation  on  43°  and  60°  agars  given  in  Fig.  B  also  show  but 
little  influence  of  these  agars  on  these  two  characters.  A  "Difco"  corn- 
meal  agar,  prepared  according  to  my  directions  by  the  Digestive  Ferments 
Company,  gave  growth-characters  almost  identical  with  those  of  my 
own  60°  agar.  On  "Difco"  beef-agar  the  conidia  were  short,  and  were 
frequently  deformed  (M,  17.44±.22,  <r,  2.46±.16,  CV,  14.15±.93). 

Plain  agar  (shredded  agar  only,  12  g.  per  liter). — The  fungus  grew 
rapidly,  and  in  6  days  the  colony  was  35-45  mm.  in  diameter,  but  was 
thin  and  colorless,  with  but  few  scattered  conidia,  and  only  1  to  3, 
or  at  the  most  7  to  10,  conidiophores  per  low-power  field,  except  at 
growth-inhibition  points,  as  at  the  edge  of  the  dish  or  where  two  colonies 
approached  each  other,  where  the  number  of  conidiophores  rose  to  about 
12  per  low-power  field.  The  conidiophores  bore  only  one  or  two  conidia 
each.  Conidiophores,  conidia,  and  mycelium  as  well,  were  very  faintly 
straw-colored,  much  paler  than  on  more  nutrient  agar.  No  zonation 
occurred.  No  difference  in  rate  or  character  of  growth  was  observable  in 
1.3%  and  2.6%  plain  agar. 

Growth  on  plain  agar,  on  corn-meal  agars,  and  on  various  combinations 
of  these  nutrients. — Corn-meal  agar  of  various  compositions  was  used, 
12  c.c.  to  each  Petri  dish.  On  25%  corn-meal  agar  (made  of  3  parts  plain 
agar  plus  1  part  ordinary  corn-meal  agar)  the  colony  was  much  darker 
and  denser  than  on  plain  agar;  was  zoned  more  strongly;  and  conidia  were 
much  more  abundant,  there  being  about  80  conidiophores  per  low-power 
field,  each  with  one  to  five  conidia.  The  mycelium  was  much  darker 
than  on  plain  agar.  Colonies  on  50%  and  75%  corn-meal  agar  showed 
no  essential  difference  from  the  colony  on  25%  agar.  On  full  corn-meal 


83 

agar  the  colony  was  much  darker  and  more  dense  and  the  mycelium  was 
darker.  The  relative  rate  of  linear  growth  on  these  agars,  as  shown  in 
millimeters  of  colony  diameter  at  the  end  of  9  days  at  room-temperature, 
was  as  follows: 


On    plain  agar 70  mm. 

On  25%  corn-meal  agar.  .68  mm. 
On  50%  corn-meal  agar.  .62  mm. 
On  75%  corn-meal  agar.  .57  mm. 
On  100%  corn-meal  agar.  .54  mm. 


When  the  fungus  was  planted  on  plain  agar,  and  pieces  (1  cm.  square) 
of  corn-meal  agar  of  the  above-mentioned  compositions  were  laid  on  the 
surface  at  the  edge  of  a  well-developed  colony,  both  color  and  conidia- 
production  increased  with  the  increase  of  nutrients.  Variation  in  conidial 
length  on  these  agars  is  shown  in  Fig.  C.  On  this  series  of  agars  conidial 
length  was  least  on  plain  agar  and  increased  consistently  with  the  strength 
of  the  medium.  It  is  to  be  noted  that  the  coefficient  of  variability  is 
very  high  on  the  75%  corn-meal  agar.  In  Graphs  9  and  10  of  this  Fig. 
C,  conidial  length  is  seen  to  be  appreciably  lower  than  on  full  corn-meal 
agar  (Graphs  1-4,  Fig.  A),  and  markedly  shorter  than  conidia  grown  under 
standard  conditions  (see  Graphs,  Fig.  K;  also  App.,  page  180).  Again,  corn- 
meal  agar  was  made  in  the  usual  way  but  the  amount  of  agar  was  varied, 
6,  12,  and  25  grams  per  liter  being  used.  In  general,  in  Petri  dishes,  12 
grams  per  liter  proved  most  suitable.  Comparisons  between  H.  No.  1 
and  H.  No.  3  on  these  three  media  showed  at  11  days  each  that  H.  No.  3 
had  grown  more  rapidly  than  H.  No.  1,  the  ratio  being  6.8:  8.5.  There 
was  usually  a  marked  difference  between  these  two  strains  on  12-gram 
corn-meal  agar,  more  marked  than  on  the  others,  H.  No.  3  showing  more 
definite  zonation  and  more  aerial  mycelium. 

In  Freudenreich  flasks,  with  the  6-gram  agar,  H.  No.  3  made  much 
aerial  mycelium  on  the  watery  surface;  H.  No.  1  made  only  a  black  pellicle 
and  no  aerial  mycelium.  On  12-gram  agar  H.  No.  1  made  small  growth 
of  aerial  mycelium  and  the  colony  surface  was  black,  while  H.  No.  3  had 
much  loose,  woolly  mycelium.  At  11  days  H.  No.  3  on  each  agar  had  more 
aerial  mycelium  and  more  clumps  than  did  H.  No.  1.  The  most  conspicu- 
ous difference  was  on  12-gram  agar,  while  on  25-gram  agar  H.  No.  1  had 
no  clumps  and  H.  No.  3  a  few. 

There  is  a  clear,  definite  tendency  in  H.  No.  3  to  make  more  aerial 
mvcelium  and  more  clumps  than  H.  No.  1,  but  this  is  so  dependent  on  con- 


84 

ditions  of  moisture,  in  air  and  medium,  that  it  is  far  from  being  a  reliable 
character  for  separating  the  two. 

Green-wheat  agar. — (Formula  as  for  corn-meal  agar,  substituting  for  the 
corn-meal  live  wheat  leaves  and  stems  which  had  been_  passed  through 
a  meat-grinder.)  H.  No.  1  grew  well,  producing  a  dense  colony,  but  with 
weak  zonation  and  with  much  woolly,  white  aerial  mycelium,  and  but  few 
and  scattered  conidia.  This  medium,  while  apparently  very  nutritious, 
favored  abundant  vegetation  rather  than  sporulation,  and  was  a  poor 
medium  for  the  differentiation  of  races. 

To  determine  the  effect  of  reducing  the  nutrients  in  green-wheat 
agar,  this  was  combined,  in  varying  proportions,  with  washed  agar  with 
varying  results.  On  washed  agar  the  growth  of  H.  No.  1  was  scant,  color- 
less, and  with  no  conidia,  the  colony  diameter  reaching  only  5.5  cm.  in 
9  days.  As  the  content  of  green  wheat  was  increased,  there  was  a  gradual 
increase  in  density  of  colony  and  of  aerial  mycelium.  In  9  days  the  colony 
diameters  were  as  follows: 


On    25%  green- wheat  agar.  .9  cm. 
On    50%  green-wheat  agar.  .8  cm. 
On    75%  green-wheat  agar.  .7.5  cm. 
On  100%  green-wheat  agar.  .7.5  cm. 


These  colonies  showed  no  dark  color  and  only  very  weak  zonation, 
and  in  the  two  high  concentrations  the  aerial  mycelium  developed  into  a 
dense,  closely  felted  mat.  Conidia  were  produced  scantily  and  varied 
greatly  from  the  shape  found  elsewhere,  being  less  tapering,  more  nearly 
cylindrical,  and  materially  thicker  (Graphs  13,  14,  Fig.  D).  In  some 
instances  septation  differed  markedly— diminished  (Graphs  15,  16,  Fig.  D). 
Many  large  conidia  had  no  septa  at  all,  and  others  had  irregular  or  incom- 
plete septa.  It  is  evident  that  this  medium,  even  at  25%  strength,  in- 
duces many  abnormalities,  and  the  very  high  coefficient  of  variability 
is  especially  striking.  The  differences  in  septation  here  noted  were  not 
constant  on  the  same  plate  and  were  much  more  common  at  the  drying 
edge.  On  this  agar  conidial  length  was  less  than  under  standard  conditions 
(see  appendix,  p.  180). 

Wheat-straw  agar. — (Fifty  grams  of  old  wheat-straw,  boiled  20  minutes 
and  filtered.)  Growth  was  poor. 

"Difco"  beef -agar. — H.  No.  1  grew  slowly  but  was  very  dense;  surface 
even;  little  aerial  mycelium. 


85 

Starch  agar. — This  medium  proved  to  be  of  but  slight  differential 
value.  The  growth  was  of  a  dark  color  and  of  somewhat  bluish  tinge. 

Bean  agar. — H.  Nos.  1,  3,  5,  13,  20,  grew  well  on  bean  agar,  all  de- 
veloping a  dense,  woolly,  gray  aerial  mycelium.  Zonation  was  poor,  or 
obscured  by  the  aerial  mycelium.  The  five  strains  showed  no  differences 
in  growth  on  this  medium,  which  was  therefore  poor  for  differential  use. 
In  Freudenreich  flasks  there  was  a  definite  black  surface-line  and  much 
tawny  aerial  mycelium  in  clumps. 

Brazil-nut  agar. — (Formula  according  to  Spencer,  108.)  H.  No.  1  in 
test-tubes  grew  very  rapidly  and  luxuriantly  with  small  development  of 
aerial  mycelium  and  a  distinct  black  basal  line.  The  agar  was  rapidly 
cleared  of  proteid  precipitate  by  the  development  of  a  proteolytic  enzyme. 
In  Petri  dishes  a  thick,  dense,  woolly,  snow-white  aerial  mycelium  devel- 
oped which  entirely  curtained  the  surface-blackening.  The  colony  was 
surrounded  by  a  broad  translucent  zone  due  to  proteolytic  action.  This 
agar  is  valuable  for  the  pure-white  aerial  mycelium  that  develops  on  it, 
and  to  demonstrate  readily  the  proteolytic  action,  though  it  did  not, 
even  in  these  regards,  prove  to  be  differential,  since  all  of  fifteen  strains 
tested  upon  it  gave  nearly  identical  responses. 

Oat  agar. — H.  No.  1  at  10  days  gave  a  distinct  black  surface-line 
and  very  heavy  aerial  gray  growth.  H.  Nos.  1,  4,  and  14  were  indistin- 
guishable on  it. 

Apple-fruit  agar. — H.  No.  1  gave  a  black  basal  line  and  abundant, 
sooty  aerial  mycelium.  H.  Nos.  1,  4,  and  14  were  alike  except  that  No.  4 
produced  large  sclerotia. 

Apple-bark  agar. — H.  Nos.  1,  4,  and  14  grew  very  slowly  and  were 
very  dense  and  black,  with  but  little  aerial  mycelium. 

Czapec  agar. — H.  No.  1  gave  a  black  surface-line  and  no  aerial  my- 
celium. H.  Nos.  4  and  14  were  of  the  same  character  except  that  No.  4 
produced  a  considerable  quantity  of  smoky  aerial  mycelium. 

Prune  agar. — H.  Nos.  1  and  4  gave  a  dense,  black  surface-growth  but 
no  aerial  mycelium. 

SUMMARY  CONCERNING  GROWTH  ON  AGARS 

Corn-meal  agar  made  by  the  usual  60°  formula  proved  most  useful,  and 
the  best  differential  agar.  If  made  at  100°  or  at  43°  it  lacked  nutri- 
ment. The  amount  of  agar  used — even  25  g.  per  1000  c.c. — had  but  little 
effect  on  growth  characters.  Green-wheat  agar  in  varying  strengths  led 
to  luxuriant  vegetation,  to  little  conidia-production,  to  much  abnormality 


86 

in  morphological  characters,  and  was  of  little  differential  value.  On 
either  washed  agar  or  plain  agar  there  was  excellent  linear  growth  but 
poor  color  and  little  conidia-production.  Bean  agar  gave  too  luxuriant 
vegetative  growth.  Brazil-nut  agar  was  useful  for  the  development 
of  white  mycelium  for  use  in  nutrition  studies  and  for  study jof^proteoly tic 
action.  The  other  agars  used  showed  no  special  features  of  value. 

RICE    AND    SIMILAR    SUBSTANCES    AS    MEDIA,    WITH    SPECIAL 
NOTE    OF    COLOR    PHENOMENA 

Rice  in  test-tubes. — Rice  was  prepared  in  the  customary  way,  by  placing 
it  in  test-tubes  to  a  depth  of  one  centimeter,  adding  enough  water  to 
stand  1  cm.  above  it,  and  then  autoclaving.  This  medium,  so  useful  in 
the  study  of  many  fungi,  notably  of  Fusarium,  proved  very  interesting 
here.  At  the  expiration  of  about  two  weeks  from  the  time  of  inoculation — 
generally  the  most  profitable  time  for  first  observation — three  zones  or 
regions  could  usually  be  recognized:  (1)  the  region  recently  invaded  by 
the  fungus,  which  I  designate  as  the  recent  region;  (2)  the  region  first 
invaded,  which  had  assumed  nearly  its  final  appearance,  and  which  I  call 
the  old  region;  and  (3)  the  region  midway  between  1  and  2,  which  I  shall 
call  the  median  region. 

Each  of  these  regions  showed  characters  of  its  own.  Within  all  of 
them,  but  particularly  in  the  old  and  in  the  median  regions,  there  were 
three  points  to  observe:  (a)  the  places  where  the  rice  grains  came  in  contact 
with  the  glass,  which  places  I  call  contact;  (b)  the  spaces  between  rice  grains, 
at  first  filled  by  water,  which  I  call  the  interstices;  and  (c)  the  line  between 
interstices  and  contacts,  which  I  term  the  border.  Usually  the  fungus 
grows  down  into  the  interstices,  consumes  their  contents,  and  fills  the 
remaining  space  more  or  less  completely  with  mycelium.  Penetration 
of  the  contacts  is  very  slow  and  may  not  occur  at  all,  therefore  they  are 
usually  but  slowly  discolored  by  the  passage  of  various  chemicals  into  them. 
The  border  is  the  region  of  greatest  development,  and  often  presents  a 
sharp,  distinct  line  of  pronounced  character.  It  is  the  contrasts  furnished 
by  the  contacts,  interstices,  and  borders,  often  attended  by  the  develop- 
ment of  beautiful  and  vivid  colors,  that  give  to  these  cultures  their  strik- 
ing appearance.  In  addition  to  these  characters  the  final  appearance  of  the 
rice  column  should  be  noted.  It  is  sometimes  digested  away  in  characteristic 
manner.  The  development  or  absence  of  sclerotia  is  also  noteworthy. 

H.  No.  1,  in  rice  test-tubes,  at  two  weeks,  gave,  in  the  recent  region, 
salmon-colored  interstices,  contacts,  and  borders;  in  the  median  regions  the 


87 

interstices  and  contacts  were  gray,  the  borders  olive;  in  the  old  regions 
the  interstices  were  dark,  contacts  gray,  and  borders  deep  black.  No 
sclerotia  developed. 

H.  No.  2  gave  quite  distinctive  color-characters,  as  did  also  Nos.  11, 
12,  16,  17,  and  19,  including  the  colors  pink,  brown,  red,  gray,  and  purple. 
The  remaining  strains  were  distinguished  only  by  sclerotial  development, 
in  which  character  strains  known  to  be  closely  related  differed  markedly. 

Additional  notes  made  on  these  cultures  at  later  periods,  up  to  four 
weeks,  differed  only  as  to  sclerotial  development  and  the  wasting  away 
of  the  rice  column.  At  four  weeks  H.  No.  1  produced  some  sclerotia,  and 
at  five  weeks,  many. 

In  an  attempt  to  ascertain  wherein  lay  the  responsibility  for  the 
various  colors  found  in  rice  cultures,  tubes  wrere  made  in  the  usual  way, 
using  rice  chaff,  whole  rice,  rice  from  the  pearling  cone,  rice  from  the 
brush,  and  rice  polish.  Of  these,  rice  chaff  and  whole  rice  gave  no  color 
reactions;  but  all  the  others  gave  the  usual  ones.  Single  grains  of  auto- 
claved  rice  wrere  placed  on  washed  agar,  under  a  cover-glass,  and  inoculated 
with  color-producing  Helminthosporiums.  Direct  microscopic  observa- 
tion showed  the  most  intense  colors  on  the  unbroken  rice-surface,  and  less 
intense  ones  on  interior  regions  exposed  by  breaking.  It  is  therefore 
probable  that  the  proteids,  which  are  most  abundant  in  the  surface  layers, 
are  necessary  to  the  color  responses,  though  the  color  itself  often,  if  not 
always,  arises  from  the  mycelium. 

Pearl-barley  (prepared  like  rice  tubes], — The  general  character  of 
the  growth  as  to  contacts,  interstices,  and  borders  was  as  on  rice  but 
wTith  different  and  less  pronounced  coloring.  H.  No.  1  at  two  weeks  gave, 
in  the  recent  region,  salmon  interstices,  contacts,  and  borders;  in  the 
median  region,  gray  interstices  and  contacts  and  olive  borders;  in  old 
regions,  dark  interstices,  gray  contacts,  and  black  borders;  and  the  aerial 
mycelium  was  gray  and  the  barley  column  not  shrunken. 

Navy  beans  (prepared  like  rice  tubes). — H.  No.  1  grown  on  this  medium 
two  weeks  gave  contacts  unchanged,  interstices  gray  to  brown,  borders 
brown  to  black.  Little  of  differential  value  developed,  though  H.  Nos.  11 
and  19  gave  pink  colors. 

Wheat  grains  (prepared  as  above). — H.  No.  1  in  old  region  gave  con- 
tacts unchanged,  interstices  dark  gray,  borders  black,  and  a  very  few 
sclerotia  developed.  H.  No.  3  gave  the  same  characters  but  more  numer- 
ous sclerotia. 


Almond  integuments  (prepared  as  above). — H.  No.  1  gave  a  dense,  black 
mycelium  and  abundant  conidia. 

Corn  meal  (moistened  and  autodaved  in  test-tubes}. — H.  No.  1  gave 
dense,  black  borders  and  greenish-black  interstices. 

Corn-starch  (prepared  as  above}. — H.  No.  1  gave  a  dense,_black,  even 
surface-growth  with  little  or  no  aerial  mycelium. 

SUMMARY    CONCERNING    GROWTH    ON    RICE    AND    SIMILAR 
SUBSTANCES 

With  Helminthosporium,  as  with  Fusarium,  rice  tubes  are  of  value  for 
differentiating  species.  The  important  constituent  seems  to  be  in  the 
aleurone  layer.  Pearl-barley  has  similar  and  different,  though  less,  value. 
Whole  wheat  grains,  whole  rice,  beans,  etc.,  do  not  give  these  color  reactions. 

Color  phenomena  in  fungi  have  been  discussed  by  Smith  (106),  and 
Hedgecock  (65),  and  by  Stewart  and  Hodgkiss  (119),  who  cite  several 
papers  bearing  upon  the  subject.  These,  however,  deal  mainly  with 
conditions  of  acidity  and  general  carbohydrates  rather  than  with  proteid 
relations.* 

MISCELLANEOUS    VEGETABLE    MEDIA 

Parts  (plugs)  of  various  vegetables  were  placed  in  test-tubes — in 
some  cases  with  glass  slips — with  about  2  c.c.  of  water  and  autoclaved. 
The  chief  resulting  characters  of  various  Helminthosporium  plantings 
on  such  media  consisted  in  the  development  of  aerial  mycelium  and 
the  pellicle  over  the  surface  of  the  water. 

Potato  plugs  (prepared  in  the  usual  way). — H.  No.  1  gave  on  these 
plugs  large  development  of  woolly,  white  aerial  mycelium.  At  the  line 
of  contact  with  the  glass  the  growth  was  dense  and  black.  No  sclerotia 
developed  in  four  days.  This  medium  possessed  little  differential  value, 
only  H.  Nos.  11,  12,  and  17  showing  clear  differences. 

Potato  plugs  on  glass  slips. — The  slips  were  placed  in  test-tubes  (in 
the  manner  shown  in  Fig.  2,  page  95),  and  then  slices  of  potato,  which  were 
so  cut  that  they  could  barely  be  crowded  into  the  tubes.  They  were  then 
autoclaved.  This  gave  opportunity  for  observation  at  three  places:  (1)  the 
exposed  potato  surface;  (2)  the  contact  of  the  potato  with  the  glass  slip  and 
with  the  wall  of  the  tube;  and  (3)  the  border  at  the  edge  of  the  contact 
(cf.  with  terms  under  rice,  p.  86).  H.  No.  1  here  gave  a  dense  black 


*In  this  connection  see  paragraph  on  carbohydrates  on  p  ^.ge  100. 


89 

border,  a  woolly,  smoky  aerial  surface,  and  was  black  at  contacts.  H. 
Nos.  11,  12,  and  17  were  otherwise  colored. 

Carrot  plugs  on  glass  slips  (manipulated  as  above). — H.  No.  1  gave 
black  border  and  scant  aerial  mycelium;  H.  Nos.  11  and  17  were  quite  dif- 
ferent. 

On  other  carrot  plugs,  placed  in  test-tubes  but  without  the  glass 
slips,  H.  No.  1  gave  at  4  days  a  slowly  developing  black  surface-growth. 
H.  No.  3  grew  faster  and  with  more  aerial  mycelium.  At  9  days,  H.  Nos.  1 
and  3  were  alike. 

Sweet-potato  plugs. — H.  No.  1  at  4  days  was  densely  woolly  on  the 
surface;  surface  of  H.  No.  3,  less  woolly.  At  9  days  H.  Nos.  1  and  3  were 
alike. 

Onion  bulbs;  radish  root. — H.  No.  1  and  H.  No.  3  had  abundant  myce- 
lium, a  pellicle,  and  conidia. 

Celery,  onion  stems,  green  peas,  pea-pods,  string-beans. — H.  Nos.  1 
and  3  had  made  dense,  white,  woolly  aerial  mycelium. 

Rhubarb  stems. — H.  No.  1  made  no  growth;  H.  No.  3,  poor  growth. 

Cranberries. — H.  Nos.  1  and  3  made  no  growth. 

Bean  broth. — H.  Nos.  1  and  3  gave  a  thick  pellicle  and  a  scant  aerial 
mycelium.  This  medium  has  little  differential  value,  though  large  white 
mycelial  clumps  appeared  in  H.  Nos.  7,  8,  and  9,  much  less  in  No.  6,  and  in 
some  cases  there  was  no  pellicle.  These  characters,  however,  appear  too 
variable  to  have  value. 

Old  wheat-straw. — Old,  dry  wheat-straw,  cut  free  of  leaves  and  into 
lengths  of  about  12  cm.,  was  placed  in  test-tubes  with  a  few  c.c.  of  water 
and  autoclaved.  Inoculated  on  these  straws,  II.  No.  1  grew  well,  largely 
covering  the  lower  part  of  the  stem  (which  was  in  the  most  humid  air) 
with  a  black  coating  of  conidia.  Each  conidiophore  usually  produced 
several  conidia.  On  the  upper  part  of  the  stem,  which  was  in  less  humid 
air,  conidia  were  produced  much  less  abundantly  and  were  usually  solitary 
on  the  conidiophore,  while  on  the  portion  of  the  stem  very  near  the  cotton 
plug  there  was  a  growth  of  aerial  mycelium.  The  fungus  also  grew  sparsely 
over  the  surface  of  the  water,  where  it  produced  conidia.  Nineteen  other 
numbers  of  Helminthosporium  were  grown  on  this  medium  and  kept 
under  observation  more  than  three  months.  Three  numbers,  11,  17, 
and  21,  showed  distinctive  characters,  and  among  the  others  there  were 
minor  differences  in  conidia  production,  in  amount  of  aerial  mycelium  on 
the  upper  end  of  the  straw,  and  in  the  density  of  the  pellicle.  These 
differences  were,  however,  as  great  between  two  cultures  known  to  be 


90 

of  the  same  species  (e.  g.,  H.  Nos.  5  and  6),  and  between  H.  No.  20  and  No. 
13,  presumably  of  the  same  species,  and  therefore  they  are  not  of  reliable 
taxonomic  value.  Even  at  the  end  of  three  months  there  were  no  sclerotia, 
pycnidia,  or  perithecia  in  any  of  the  tubes.  Ravn  (91)  has  stressed  the 
importance  of  sclerotia  (which  this  medium  yielded  in  his  jstudies)  as  a 
means  of  distinguishing  certain  races  or  species;  but  no  such  value  attaches 
to  it  for  the  races  that  I  had  under  observation. 

Fresh  wheat-leaves. — Green  wheat-leaves  were  prepared  in  the  same 
manner  as  the  wheat  straw.  On  these  leaves  growth  of  H.  No.  1  was 
much  as  on  the  wrheat  straw  except  that  many  more  conidia  were  pro- 
duced both  on  the  lower  portions  of  the  leaf  and  on  the  water-surface. 
On  the  upper  part  of  the  leaf,  about  6  cm.  above  the  level  of  the  water, 
where  the  leaf  was  too  dry  for  conidia-formation,  a  rather  extensive,  white, 
floccose,  loose  aerial  mycelium  developed. 

SUMMARY  CONCERNING  THE  FOREGOING  VEGETABLE  MEDIA 

"But  little  of  differential  value  resulted.  The  characters  of  aerial 
mycelium,  sclerotia,  clumping  of  mycelium,  and  pellicle-formation  were 
but  slightly  marked,  all  being  highly  dependent  on  conditions  of  environ- 
ment. 

CEREAL    SHOOTS    GROWN    FROM    ASEPTIC    SEEDS    AS    MEDIA 

Cereal  seeds  were  disinfected  and  sprouted  in  sterile  moist  cham- 
bers. When  the  shoots  were  2-3  cm.  long  they  were  cut  off,  placed  in 
water  in  a  test-tube,  and  autoclaved.  Next,  washed  agar  was  placed  in 
Petri  dishes  and  inoculated  with  H.  No.  1.  About  four  days  later,  when 
the  colonies  were  several  centimeters  in  diameter,  various  cereal  shoots, 
prepared  as  above,  were  laid  on  the  washed-agar  plates,  the  basal  end  of 
the  shoot  in  each  case  touching  the  edge  of  the  colony.  In  this  way  re- 
peated tests  were  made  writh  wheat,  oats,  corn,  rye,  and  barley,  with  the 
invariable  result  that  growth  was  most  luxuriant  and  dense  on  the  corn, 
which  soon  became  completely  black.  No  constant  difference  was  evident 
between  the  other  cereals,  except  that  rye  seemed  a  medium  slightly  less 
favorable  than  the  others. 

It  seemed  probable  that  the  more  luxuriant  growth  and  conidia-devel- 
opment  on  the  corn  shoots  was  due  to  quantity  rather  than  to  quality  of 
nutrients.  To  test  this  hypothesis  an  entire  corn  shoot,  a  longitudinal 
half  of  a  shoot,  a  longitudinal  quarter  of  a  shoot,  and  a  mere  longitudinal 
filament  were  laid  on  a  colony  of  H.  No.  1  growing  on  washed  agar.  On 
the  smallest  fragment,  growth  and  conidia-production  were  about  as  on 


91 

wheat.  The  quantity  of  conidia  produced,  and  correspondingly  the  black 
color,  bore  a  direct  relation  to  the  mass  of  the  corn  shoot.  It  is  probable 
that  all  of  these  autoclaved  shoots  are  of  equal,  or  nearly  equal,  nutritive 
value  for  a  given  amount  of  material. 

Eleven  other  strains  of  Helminthosporium  were  tested  in  similar  man- 
ner, using  wheat,  oat,  and  barley  shoots.  No  significant  differences  in 
growth  appeared,  though,  in  most  cases,  at  the  end  of  the  growth-period 
there  was  a  somewhat  more  profuse  development  of  conidia  on  wheat  than  on 
the  two  other  cereals. 

ENVIRONMENTAL  FACTORS  WHICH  INDUCE  VARIATION 

Since  classification  of  these  fungi  is  necessarily  based  primarily  on 
morphology,  and  on  growth  characters  as  exhibited  on  various  media, 
it  is  important  to  know  what,  and  how  great,  variations  in  these  charac- 
ters are  induced  by  environmental  changes.  It  is  generally  conceded  that 
culture  characters  to  be  comparable  must  be  noted  under  similar  condi- 
tions. But  how  much  latitude  is  permissible  here;  what  difference  in 
environment  may  be  regarded  as  negligible?  Fungi  are  collected  that  have 
grown  under  different  conditions  of  nutrition,  humidity,  temperature,  etc., 
and  specific  descriptions  are  written,  based  on  these  collections.  To 
what  extent  are  they  trustworthy?  The  size  and  shape  of  the  conidia  and 
septation  are  regarded  as  of  the  highest  taxonomic  value.  The  develop- 
ment of  sclerotia,  of  aerial  mycelium,  of  mycelial  clumps,  of  color,  etc., 
are  often  the  only  characters  separating  some  types  regarded  as  species. 
Even  the  characters  of  the  colony  on  the  agar  plate  are  considered  im- 
portant. It  is  with  the  hope  of  throwing  some  light  upon  this  phase  of 
the  subject  that  I  record  the  following  additional  observations  concerning 
environmental  factors  which  induce  variation.  More  complete  and  ex- 
haustive studies  on  these  and  kindred  phenomena  are  needed. 

QUANTITY    OF    NUTRIMENT 

Petri  dishes  were  respectively  supplied  with  12  c.c.  and  30  c.c.  of 
60°  corn-meal  agar  and  inoculated  with  H.  No.  1.  Placed  under  the 
same  conditions,  these  dishes  gave  very  different  colony-characters. 
The  colonies  on  the  plates  containing  the  larger  amount  of  agar  were  very 
dense  and  black,  and  covered  the  agar-surface  much  more  completely  than 
did  the  colonies  on  the  plates  containing  less  agar  (PI.  XIV).  There  was 
also  marked  difference  in  the  rate  of  linear  growth,  as  is  shown  in  the  fol- 
lowing table: 


92 


age 

At 

11  days1  age 

30  c.c.  of  agar 

On  12  c.c.  of 

agar       30  c.c.  of  agar 

pi.    8,  32 

pi.  1,  60 

pi.    8,  85 

pi.    9,40 

pi.  2,  65 

.  pl._9.  90 

pi.  10,  32 

pi.  3,  65 

pi.  10,  85 

pi.  11,  40 

pi.  4,  63 

pi.  11,  85 

pi.  12,  35 

pi.  5,  63 

pi.  6,  62 

•pi.  7,  65 

Totals,   179 

Totals,  443 

Totals,  345 

Av.  per  pi., 

Av.  per  pi 

.,                  Av.  per  pi., 

35.8  mm. 

63.3  mm 

86.2  mm. 

GROWTH    IN    DIAMETER    OF   COLONIES    (MILLIMETERS) 


On  12  c.c.  of  agar 
pi.  1,  22 
pi.  2,  25 
pi.  3,  29 
pi.  4,  32 
pi.  5,  25 
pi.  6,  29 
pi.  7,  27 

Totals,  189 
Av.  per  pi., 
27  mm. 

It  appears  that  at  4  days  the  colonies  on  the  deep  agar  grew  more 
than  32%  faster  than  those  on  the  plates  with  less  agar;  at  11  days,  more 
than  28%  faster,  the  colonies  being  at  the  same  time  more  dense.  A 
repetition  of  the  test  with  16  plates  gave  at  9  days  a  growth-average  of 
50  mm.  on  the  shallow  agar  and  of  74  mm.  on  the  deep  agar — an  increase 
of  48%  in  rate. 

Since  it  was  deemed  possible  that  the  differences  here  noted  might 
be  due  to  differences  in  humidity,  the  depths  of  several  plates  were  care- 
fully measured  and  sufficient  agar  poured  into  each  to  make  the  air-space 
above  the  agar  in  all  cases  equal,  although,  since  half  of  the  plates  were 
shallow  and  half  of  them  deep,  there  was  a  great  difference  in  the  depth 
of  agar  used,  which  averaged  in  the  deep  plates  about  11  mm.  and  in  the 
shallow  plates  about  3  mm.  The  results  of  this  test  were  almost  identical 
with  those  recorded  above.  The  differences  noted,  therefore,  could  hardly 
have  been  due  to  differences  in  humidity,  but  rather  to  the  amount  of 
nutriment  available.  The  graphs  of  conidial  length  given  in  Fig.  E  show  a 
larger  predominance  of  short  conidia  on  the  deep  agar,  indicating  the  contin- 
uance of  conidia-formation  for  a  longer  period  of  time.  For  this  reason 
the  mean  length  is  much  lowered  and  the  coefficient  of  variability  on  the 
deeper  agars  is  much  higher  than  on  the  plates  with  less  agar. 

These  results  on  deep  agar  are  in  entire  agreement  with  those  already 
given  concerning  corn  shoots  (page  90),  to  the  effect  that  quantity  of  nutri- 
ment available  may  influence  the  growth-characters  very  markedly,  often 
as  distinctly  as  does  the  quality  of  the  food  available.  The  dilution  ex- 
periments with  green-wheat  agar  and  corn-meal  agar  (pages  83  and  84)  tend 
toward  the  same  conclusion,  though  other  factors  also  appear  in  those  cases. 


93 

INHIBITORY    INFLUENCES 

Many  influences  which  inhibit  or  retard  vegetative  growth,  in  so  doing 
call  forth  increased  sporulation,  in  accordance  with  the  second  law  of 
Klebs  ( 74)  that  the  conditions  favoring  sporulation  are  always  more  or  less 
unfavorable  for  growth.  Thus,  two  colonies  of  H.  No.  1  on  washed  agar 
were  independently  nearly  devoid  of  conidia;  but  when  they  grew  and  ap- 
proached each  other,  vegetative  growth  was  retarded,  and  eventually  in- 
hibited, and  each  colony  became  dark  in  the  region  of  inhibition,  owing  to 
much-increased  sporulation  (Fig.  1).  Colonies  of  many  other  species  of 
fungi  affected  H.  No.  1  similarly  under  like  circumstances,  as  did  also  dry- 
ing out  of  the  agar  at  the  colony's  edge.  Similar  changes  in  growth,  and 
consequently  in  colony-character,  occur  on  almost  any  medium,  prom- 
inently on  corn-meal  agar,  and  they  must  be  understood  and  reckoned 
with  if  colony  characters  are  to  be  used  for  descriptive  purposes. 


Fig.  1  — Illustrating  inhibitory  influence  on 
sporulation.  Two  colonies  of  H.  No.  1,  on  washed 
agar,  showing  dark  bands,  due  to  abundant  sporu- 
lation where  the  colonies  approach  each  other. 
Sporulation  also  somewhat  increased  near  margins 
of  colonies,  owing  to  drying. 


HUMIDITY    OF    MEDIA 


Rice  was  placed  in  tubes  with  water  equal  to  twice  the  volume  of  the 
rice,  and  with  water  equal  to  four  times  its  volume,  being  then  autoclaved 
and  inoculated  with  various  Helminthosporiums.  Notes  at  two  weeks 
show  better  characters  of  border,  interstices,  etc.,  in  the  wetter  tubes,  and 
while  with  many  strains  sclerotia  developed  abundantly  in  the  drier  tubes 


94 

they  did  not  appear  at  all  in  the  wetter  ones  (PI.  XV).  This  was  notably 
true  of  H.  Nos.  1  and  5.  Even  at  the  end  of  five  weeks  no  sclerotia  were 
produced  in  the  wet  tube  by  H.  No.  1.  It  is  apparent  that  the  smaller 
amount  of  water  is  the  more  favorable  for  observation  olsjclerotiu in- 
formation, either  at  two  or  four  weeks,  and  that  observation  of  wet  tubes 
at  the  end  of  two  weeks  is  sufficient  for  other  characters.  Ravn  (91)  has 
suggested  sclerotial  formation  as  a  character  for  the  separation  of  certain 
species.  It  is  obvious  that  if  this  character  is  employed  care  must  be  given 
to  the  humidity  of  the  media. 

HUMIDITY    OF    AIR 

Test-tubes  were  prepared  with  water  in  the  bottom  and  a  glass  -lip 
inserted  as  is  shown  in  Fig.  2,  and  autoclaved.  If  filter-paper  or  a  wheat 
leaf  is  laid  on  the  glass  slip  the  humidity  is  sustained  throughout  the  length 
of  the  tube  by  evaporation  from  the  leaf  or  the  paper,  but  if  no  such  con- 
ductor of  water  is  used,  and  cereal  (e.g.  wheat)  shoots  are  laid  crosswise 
on  the  glass  slip  (as  indicated  in  Fig.  2)  and  inoculated,  the  growth  of  the 
fungus  may  be  observed  under  different  conditions  of  air-humidity. 

Determinations  kindly  made  for  me  by  Dr.  G.  L.  Peltier  with  espe- 
cially accurate  apparatus  devised  by  Dr.  C.  F.  Hottes,  showed  that  moist 
wheat  sprouts  became  shriveled  and  apparently  dry  in  3  hours  at  a  relative 
humidity  of  0  to  60%;  in  12  hours,  at  70%  and  80%;  in  24  hours  at  90%, 
and  that  only  in  a  relative  humidity  greater  than  90%  did  the  sprouts 
remain  apparently  moist  for  a  longer  time. 

Test  1.  Moist  sterile  wheat-shoots  were  placed  on  the  glass  slip  (as 
shown  in  Fig.  2)  with  centimeter  intervals  between  them,  and  inoculated 
with  Helminthosporium  No.  1.  All  shoots  3  cm.  above  the  water-level 
dried  within  24  hours,  indicating  that  a  relative  humidity  as  high  as  90% 
existed  only  in  the  region  of  the  lowest  shoot  and  that  next  above  it.  In  the 
region  of  approximately  90%  relative  humidity  many  of  the  conidiophores 
became  abnormally  long  (600  ju),  and  the  basal  part  was  of  mycelial  rather 
than  of  conidiophore  character  (cf.  Fig.  3) .  It  was  apparent  from  observa- 
tions on  shoots  in  these  various  humidities  that  when  the  air  was  too  dry 
for  conidia-production  there  was  a  considerable  development  of  aerial  my- 
celium, which  accounts  for  the  fact  that  frequently  the  basal  portion  of  a 
vegetable  stem  (e.  g.,  celery  or  wheat)  may  bear  conidia,  while  the  upper 
part  bears  a  tuft  of  woolly  aerial  mycelium. 

Test  2.  Old,  dry  wheat-straw  with  water  in  test-tubes  was  autoclaved 
and  inoculated  with  H.  No.  1.  In  the  humid  bottom  the  conidia-clusters 


95 


were  large,  as  was  also  each  conidium.  Toward  the  dry  end  the  conidia- 
clusters  and  the  conidia  both  became  smaller.  From  graphs  of  conidial 
length  (Fig.  F)  it  is  obvious  that  the  mean  length  of  the  conidium  is  much 


FIG.  2. — Showing  at  left,  glass  slip  in  test-tube:  0  =  water  level, 
1,2,3,  etc.,  position  of  wheat  shoots.  In  other  test-tube,  combustion 
boat  supported  on  a  bit  of  glass  tubing,  colonies  growing  on  the  agar 
in  the  boat. 


96 


FIG.  3.— H.  No.  21,  showing  origin  of  conidiophores  from  mycelium 
under  humid  conditions,  the  conidiophores  being  very  short,  thick 
crooked,  and  black. 


97 

reduced  by  an  environment  of  relatively  dry  air;  also  that  the  relative  vari- 
ability is  much  increased  by  these  conditions  (cf.  data  of  Fig.  F  with  data 
of  Fig.  A  and  Fig.  G) .  The  great  influence  of  humidity  on  the  morphology 
of  conidia  and  conidiophores  is  discussed  by  Wartenweiler  ( 124) .  Pammel, 
King,  and  Bakke  show  by  respective  illustrations  (90,  PI.  I,  figs.  1,2,3,  and 
13-16)  large  variation  in  shape  and  size,  and  apparently  in  mode,  of  conidia 
of  Helminthosporium  as  grown  in  the  field  and  in  the  greenhouse.  Similar 
effects  from  humidity  were  noted  by  Beach  (12)  in  Septoria.  The  relative 
paucity  of  sporulation  and  the  tendency  of  H.  No.  1  to  turn  to  the  produc- 
tion of  aerial  mycelium  unless  the  air-humidity  was  very  high,  and  to  pro- 
ceed to  profuse  conidia-formation  only  when  the  relative  humidity  was 
above  90%,  explains  the  failure  to  find  Helminthosporium  conidia  on  dis- 
eased wheat-stems  in  ordinary  moist-chamber  conditions,  though  these 
same  stems,  when  given  very  moist  conditions,  invariably  became  covered 
with  Helminthosporium  conidia. 

The  six  strains  of  Helminthosporium  subjected  to  the  humidity  tests 
all  agreed  in  essential  behavior  and  characters  with  the  reaction  of  H.  No.  1 
to  the  same,  as  given  above;  but  there  is  apparent  disagreement  here  with 
the  conclusions  of  Ravn  (91)  who  says  that  he  placed  "no  water  in  the  damp 
room  (Boettcher  chamber)  since  the  moisture  would  be  so  great  that  it 
would  prevent  the  development  of  conidia." 

Test  3.  Under  a  bell  jar  lined  with  filter-paper  dipping  in  water — 
thus  securing  a  close  approximation  to  a  saturated  atmosphere — open 
Petri-dishes  of  inoculated  corn-meal  agar  were  placed.  Under  these  con- 
ditions H.  No.  3  made  much  more  white  aerial  mycelium  than  did  H. 
No.  1,  and  also  grew  faster,  whether  on  thin  (30  c.c.)  or  thick  (60  c.c.) 
layers  of  agar. 

It  here  appears  that  a  condition  of  excessive  humidity  best  develops  the 
differential  characters  of  the  aerial  mycelium  of  these  closely  related  races. 

Test  4.  H.  No.  1  was  grown  on  corn-meal  agar,  and  when  the  colony 
was  well  developed  it  was  dried  slowly  until  growth  completely  stopped. 
This  resulted  in  a  dense  black  band  of  conidia  near  the  edge  of  the  colony. 
There  were  many  conidia  upon  each  conidiophore,  as  many  as  thirteen  being 
counted  in  one  instance,  and  the  conidia  clusters  resembled  bunches  of 
grapes.  Only  the  oldest  conidia  of  a  cluster  were  at  all  large  and  these  fell 
far  below  the  mean  as  grown  under  standard  conditions,  while  the  other 
conidia  were  much  below  the  usual  size  (cf.  Graphs  62-64  [Fig.  Ol  and  39-42 
[Fig.  Kj)  and  extremely  variable,  with  coefficient  of  variability  29.99  as 
compared  with  12.22  under  standard  conditions  (Graph  42,  Fig.  K).  It 


98 

is  obvious  that  such  drying  as  is  here  recorded  results  in  increase  in  the 
number  of  conidia  per  conidiophore;  in  great  reduction  in  the  modal  mean 
and  length;  in  increase  in  variability;  and  even  in  bimodality  of  length- 
graph.  A  count  of  scars  per  conidiophore  gives  the  following:  1  case  with 
4  scars ;  1  with  5 ;  3  cases  with  6  scars ;  3  with  7 ;  2  with  8 ;  2  witrr9rand  1  case 
with  10.  This  record  affords  a  marked  contrast  with  the  more  usual  con- 
dition of  only  1,  2,  or  3  conidia  per  conidiophore. 

TEMPERATURE    RELATIONS 

The  following  is  the  record  of  H.  No.  1  on  10°,  15°,  25°,  and  30° 
washed  agar  at  87  hours:  at  10°,  trace  of  growth;  at  15°,  colony  10  mm.  in 
diameter;  at  25°,  21  mm.  in  diameter,  at  30°,  19  mm. 


GROWTH  OF  H.  No.  1  ON  CORN-MEAL  AGAR 


Temper- 
ature 

Dish 
No. 

Growth  of  colony-diameter  (millimeters)  at  time-periods  indicated 

2  da. 

3  da. 

4  da. 

5  da. 

6  da. 

7  da. 

9  da. 

13  da. 

17  da. 

20  da. 

10° 

1 

4 

11 

15 

18 

22 

29 

40 

49 

55 

2 

7 

12 

16 

20 

27 

39 

48 

56 

Average 

5.5 

11.5 

17 

21 

28 

39.5 

48.5 

55.5 

15° 

1 

8 

18 

28 

39 

50 

61 

76 

2 

10 

21 

31 

39 

48 

55 

67 

Average 

9 

19.5 

29.5 

39 

49 

58 

71.5 

20° 

1 

16 

28 

41 

53 

63 

73 

92 

2 

13 

27 

39 

51 

60 

69 

87 

Average 

14.5 

27.5 

40 

52 

61.5 

71 

89.5 

25° 

1 

17 

34 

48 

61 

75 

90 

2 

19 

34 

48 

60 

74 

90 

Average 

18 

34 

48 

60.5 

74.5 

90 

30° 

1 

18 

32 

45 

57 

71 

84 

2 

20 

34 

46 

57 

72 

85 

Average 

19 

33 

45.5 

57 

71.2 

84.5 

Steady  increase  in  growth-rate  is  apparent  up  to  25°  with  a  slight 
decrease  at  30°.  Bakke  (6)  places  the  optimum  for  H.  teres  as  23° — 
25°.  Various  changes  in  the  appearance  of  the  colony  resulted  from  cer- 
tain temperatures.  Thus  at  10°  there  was  no  zonation,  no  aerial  mycelium, 
and  very  few  conidia.  At  15°,  zonation  was  very  faint,  barely  perceptible; 
at  20°  and  25°  more  marked;  while  at  30°  the  aerial  mycelium  was  very 
scant  and  zonation  lessened. 


99 


Ravn  (91)  places  the  upper  limit  for  growth  at  33° — 34°;  the  optimum 
at  25°— 30°;  the  lower  limit  at  3° — 5°, 

Plates  prepared  in  a  similar  manner  to  the  above  were  kept  for  4  days 
at  15  °  and  then  transferred  to  the  25°  case.  Similarly,  transfers  were 
made  from  the  25°  to  the  15°  case.  The  data  of  colony-growth  from  this 
trial  are  shown  in  the  following  table. 

GROWTH  OF  H.  No.  1  IN  ALTERNATING  TEMPERATURES 


Change  of 

Temperature 

2  da. 

3  da. 

4  da. 

temperature 

5  da. 

6  da. 

7  da. 

9  da. 

13  da. 

to 

8 

18 

31 

44 

58 

72 

90 

15° 

9 

19 

28 

25° 

39 

55 

69 

90 

Average     .... 

8.5 

18.5 

29.5 

41.5 

56.5 

70.5 

90 

17 

32 

48 

55 

64 

69 

77 

90 

25° 

18 

31 

47 

15° 

54 

63 

71 

78 

90 

Average 

17  5 

31  5 

47.5 

54.5 

63.5 

70 

77  5 

90 

The  transfer  to  different  temperatures  made  no  perceptible  difference 
in  colony-character  other  than  differences  dependent  on  the  rate  of  growth. 

To  ascertain  the  relation  of  temperature  to  the  growth  of  H.  No.  1 
on  live  wheat  shoots,  this  organism  was  planted  as  under  standard  conditions 
(App.,  page  180),  except,  of  course,  that  the  shoots  were  not  autoclaved. 
Three  temperatures,  15°,  20°,  and  30°  were  used.  When  growth  had  pro- 
ceeded to  completion  conidial  length  was  found  to  be  as  shown  in  graphs  of 
Fig.  G.  Conidia-production  was  scant  at  30°,  permitting  only  a  few  meas- 
urements. At  the  other  temperatures  it  appeared  to  be  normal.  On  these 
live  shoots  there  was  a  marked  shifting  from  the  mean  to  the  lower  length 
as  we  passed  from  15°  to  20°,  and  a  still  more  marked  shifting  at  30°  (see 
Fig.  G). 

LIGHT 

Two  Petri  dishes  of  corn-meal  agar  were  inoculated  with  H.  No.  1  and 
kept  in  the  dark  at  25°.  In  the  following  table  the  resulting  growth  is 
compared  with  that  of  similar  plates  kept  in  the  light. 

In  darkness  there  was  slightly  less  zonation  and  less  aerial  mycelium 
than  in  the  light,  but  the  difference  was  only  slight  and  is  probably  insig- 
nificant. 

To  ascertain  whether  plate-position  is  an  environmental  factor  of 
importance,  numerous  corn-meal  agar  plates  were  placed  top  up,  others 


100 


bottom  up,  and  observed  carefully,  but 
of  growth  was  observed. 


GROWTH  OF  H.  No.  1  IN  THE  DARK  AND  IN  THE  LIGHT 


Temperature 

Condition 

Growth  of  colony-diameter  (millimeters)  at 
time-periods  indicated 

2  da. 

3  da. 

4  da. 

5  da. 

6  da. 

7  da. 

25° 
Average 

Dark 

21 
21 
21 
17 
19 
18 

32 
33 
32.5 
34 
34 
34 

44 
46 
45 

48 
48 
48 

56 
59 
57.5 
61 
60 
60.5 

71 
74 
72.5 
75 
74 
74.5 

85 
85 
85 
90 
90 
90 

25° 
Average 

Light 

CARBOHYDRATES 

Brazil-nut  agar  in  Petri  dish  with  H.  No.  1  gave  snowy  white  colonies. 
When  such  colonies  had  reached  a  diameter  of  about  3  cm.  one  oese  of 
powdered  dextrin,  maltose,  rhamnose,  or  glucose  was  placed  on  the  agar 
a  few  millimeters  from  the  advancing  edge  of  the  colony.  Within  48  hours 
the  portion  of  the  colony  near  the  added  carbohydrate,  with  the  exception 
of  rhamnose,  produced  conidia  in  much  greater  abundance  than  before,  and 
the  mycelium  turned  slightly  dark.  Starch,  corn-oil,  and  corn-meal  pro- 
duced a  similar  effect,  but  with  delay  of  nearly  48  hours,  suggesting  that 
the  additional  time  required  was  needed  for  the  production  and  action  of 
enzymes,  diastase,  or  lypase,  as  the  case  might  be. 

Again,  plain-agar  plates  were  poured  and  inoculated  with  H.  No.  1, 
and  when  the  colony  was  well  developed  various  carbohydrate  nutrients 
were  laid  on  the  agar  near  the  advancing  edge  of  the  colony:  steamed  rice, 
steamed  tapioca,  1  square  centimeter  of  standard  corn-meal  agar,  a  frag- 
ment of  a  Brazil-nut,  corn-starch,  wheat  flour,  corn-meal,  and  buckwheat 
flour.  All  of  these  nutrients  were  used,  and  in  each  case  the  colony  in  the 
region  of  these  nutrients  turned  black,  owing  to  the  quantity  of  conidia  pro- 
duced (PI.  XVI).  Ravn  (91)  noted  a  distinct  relation  between  carbohy- 
drate nutrients  and  blackness  in  Helminthosporium,  as  did  I  in  various 
fungi  (118). 

On  plain  agar,  glycocoll  and  aspartic  acid  inhibited  strongly  at  first, 
but  later  the  fungus  grew  through  these.  It  grew  normally  through  ty- 
rosine,  glutamic  acid,  leucin,  cystine,  phenyl,  and  alanine  without  percep- 


101 

tible  influence  £si  tfe  dumber -of  eonidia,  though  as  it  approached  corn-meal 
agar  conidia-production  became  profuse. 

NUTRIENTS  AS  AFFECTING  CONIDIAL  LENGTH,   SEPTATION,  AND  SHAPE 

Plates  of  washed  agar  when  solid  were  inoculated  writh  H.  No.  1,  and 
when  the  colony  had  grown  to  a  diameter  of  about  3  cm.  one  of  various 
nutrients  was  laid  on  the  agar  in  approximately  equal  volume,  at  a  distance 
of  1  cm.  from  the  edge  of  the  colony.  When  growth  had  ceased,  graphs  of 
conidial  length  were  made.  These  graphs,  with  data  sufficiently  explana- 
tory, are  given  in  Fig.  H.  While  the  number  of  measurements  made  is  too 
small  to  warrant  any  definite  conclusion  as  to  nutritive  values,  the  obvious 
general  conclusion  is  that  the  added  nutrient  did  markedly  affect  conidial 
length.  It  is  particularly  noticeable  that  washed  agar  plus  saccharose, 
tapioca,  or  rice  gave  small  conidia,  and  in  none  of  these  cases  was  modal 
conidial-length  equal  to  that  of  conidia  grown  under  standard  conditions 
(see  Fig.  K).  Even  the  very  striking  modification  represented  by  the  bi- 
modal  curve  shown  in  Fig.  I  was  a  product  of  environmental  change.  It 
was  noted  in  the  sample  from  which  the  graph  was  plotted  that  the  conidia 
were  produced  in  rather  large  clusters,  the  oldest  one  being  largest,  the  others 
mostly  much  smaller.  The  minor  mode  here  apparently  represents  conidia 
in  a  stage  of  arrested  development,  comparable  with  those  of  Graph  62 
(Fig.  O),  while  the  major  mode  stands  for  conidia  that  approached  more 
nearly  to  normal  development  but  did  not  attain  full  size  (cf.  graphs  of 
Figs.  I  and  O  with  those  of  Fig.  K).  That  the  bimodality  is  not  due  to 


FIG.  4. — Tri-pointed  conidia   of  I  H.  No. 
23  and  H.  No.  36  (see  text.  p.  102). 


102 

saltation  is  clear,  since  transfers  made  from  these  plates  to  wheat  shoots 
(standard  conditions;  see  App.,  p.  180)  gave  normal  curves. 

A  striking  temporary  modification  of  conidial  shape,  due  presumably 
to  nutritive  or  osmotic  conditions,  was  exhibited  by  H.  Nos^^S,  and  36, 
in  which  a  considerable  number  of  the  conidia  were  tri-pointed,  owing  to 
the  central  cell  becoming  inequilateral,  then  extending  as  is  shown  in  Fig. 
4.  The  fungus  had  long  been  cultured  on  rich  carbohydrate  agar,  and 
when  transferred  to  corn-meal  agar  these  peculiar  tri-pointed  conidia  were 
no  longer  common.  (In  this  connection,  see  also  page  154.) 


SUMMARY    CONCERNING    ENVIRONMENTAL    FACTORS    WHICH 
INDUCE    VARIATION 

It  is  obvious  from  the  foregoing  record  that  very  slight  changes  in  en- 
vironment may  produce  marked  alterations  in  colony  characters — growth- 
rate,  color,  aerial  mycelium,  clumping  of  mycelium,  and  even  in  the  shape, 
septation,  and  size  of  the  conidia.  The  quantity  and  concentration  of 
nutrients,  particularly  of  proteids  and  carbohydrates,  have  a  very  import- 
ant influence  on  conidia-production  and  colony-color.  Steinberg  (114)  and 
Javillier  (69)  have  shown  that  even  the  zinc  in  the  glass  of  the  culture- 
flask  has  marked  effect  on  the  culture-characters.  Dastur  (40)  has  noted 
the  following  effect  of  media  on  Gloeosporium:  after  long  culture  on  agar 
the  conidia-bearing  capacity  is  lost,  though  return  to  favorable  media  leads 
to  its  recovery  unless  the  stay  on  agar  has  been  too  prolonged,  in  which 
case  the  capacity  to  bear  conidia  is  completely  lost.  Air-humitidy  is  es- 
pecially significant  as  regards  aerial  mycelium,  and  has  also  an  appreciable 
influence  on  the  size  of  the  conidia.  Light  seems  of  less  importance,  though 
experiments  were  insufficient  with  this  factor  to  be  conclusive.  Tempera- 
ture in  the  upper  and  lower  ranges  has  a  marked  influence  on  many  mor- 
phological characters. 

In  the  light  of  the  foregoing  experiments  it  is  clear  that  comparisons  of 
colony-characters  on  artificial  media  should  be  made  (especially  as  regards 
points  of  minor  difference)  only  on  media  known  to  be  as  nearly  as  possible 
of  the  same  composition,  and,  when  practicable,  upon  the  same  lot  of  medi- 
um and  at  the  same  time;  thus  only  can  truly  parallel  conditions  be  secured. 
Petri  dishes  with  flat  bottoms  should  be  used,  and  in  order  to  secure  equal 
thickness  of  medium  the  agar  should  solidify  when  level.  Straw,  leaves, 
etc.,  in  test-tubes,  give  different  quantities  of  aerial  mycelium  and  conidia 
of  different  size  at  different  levels,  both  of  which  are  due  to  humidity  and 


103 

nutrition  differences.  It  is  clear  that  conidia  developed  in  the  open  on 
leaves  or  stems — some  of  the  conidia  in  humid  conditions,  some  in  dry; 
some  on  leaves  of  rich  nutrient  value,  others  on  those  less  nutritious — may 
differ  materially  in  measurement  owing  to  environmental  differences;  and 
similarly,  it  is  clear  that  sclerotium-development  is  markedly  influenced  by 
slight  changes  in  humidity. 

Ravn  (91)  states  that  conidia  measured  from  one  plant  were  thicker 
than  those  from  another,  but  whether  this  was  due  to  environment  or  to 
actual  differences  in  the  fungi  was  not  stated.  He  does,  however,  state 
definitely  that  in  his  groups  conidia  collected  under  different  conditions 
showed  different  lengths;  and  that  their  length  depends  upon  the  conditions 
under  which  they  developed.  Examples  of  extensive  modifications  due 
to  substratum  are  mentioned  by  Edgerton  (50),  Coons  (33),  Duggar  (45), 
Moreau  (84),  and  Burger  (32).  Variations  similar  to  those  herein  noted 
as  due  to  substratum,  especially  those  due  to  the  relation  of  carbohydrate 
nutriment  to  color  and  alteration  of  conidial  mode  were  noted  by  me  in 
1909  (Stevens  and  Hall,  118).  Brierley  (26)  denominates  such  variation 
"modal  variation." 

MORPHOLOGY  OF  THE  FOOT-ROT  FUNGUS 

The  parts  chiefly  to  be  considered,  in  the  absence  of  knowledge  of  the 
ascigerous  stage,  are  the  mycelium,  aerial, surface, and  submerged,  conid- 
iophores,  and  conidia.  Discussion  of  the  relation  of  the  mycelium  in  and 
to  host-tissue  will  be  given  under  the  heading  "Infection  phenomena 
on  wheat"  (page  128). 

This  morphological  study  was  made  in  part  on  the  various  media  here- 
tofore mentioned  and  on  diseased  plants  in  moist  chamber.  It  was  impor- 
tant, however,  to  have  some  means  of  securing  the  conidia,  and  other  or- 
gans above  specified,  in  large  quantity,  in  form  readily  and  conveniently 
available  for  examination.  Moreover,  the  variability  of  the  fungus  under 
slight  environmental  changes  emphasized  the  necessity  of  securing  such 
morphological  units  as  had  developed  under  conditions  as  nearly  identical 
and  uniform  as  possible.  This  led  to  search  for  some  standard  means  of 
culturing  the  conidia.  Evidently  corn-meal  agar  was  unsatisfactory,  since 
very  slight  changes  in  quality  due  to  mode  of  preparation  led  to  great  mor- 
phological changes.  Green-wheat  agar  and  bean  agar,  both  open  to  the 
same  objection,  also  gave  too  much  vegetative  growth  and  too  few  and  often 
abnormal  conidia.  Wheat  in  moist  chamber,  or  wheat  leaves  or  shoots  in 
test-tubes  varied  so  much  in  humidity  that  they  could  not  give  constant 


104 

morphological  characters.     The  procedure  finally  adopted  to  secure  stand- 
ard conditions  may  be  found  in  the  appendix,  page  180. 


FIG.  5. — H.  No.  1.  showing  extensive  anastomosis  of  the  mycelium  where  it  coursed  over  bare 
glass:  a,  low- power  view,  b,  detail  of  portion  of  a,  high  power;  c,  a  bit  of  ropy  mycelium  com- 
posed of  several  twisted  hyphae,  many  of  the  single  threads  undergoing  dissolution. 


105 

MYCELIUM 

The  young,  vigorous  mycelium  growing  within  the  agar  is  smooth, 
quite  straight,  nearly  or  quite  hyaline,  and  of  very  uniform  diameter.  The 
general  aspect  near  the  edge  of  a  colony  is  shown  in  Fig.  6,  d.  In  older 
parts  of  the  colony  the  submerged  mycelium  is  somewhat  thicker  and  is 
less  rich  in  protoplasm.  The  mature  to  old  submerged  mycelium  has  in 
agar  a  barely  perceptible  tinge  of  straw-color.  The  cells  are  quite  strongly 
constricted  at  the  septa  and  the  content  is  highly  vacuolate.  Ravn  (91) 
states  that  in  the  old  immersed  mycelium  both  plasma  and  cell-walls  may  be 
blackish  green,  grayish  brown,  or  entirely  black  as  in  Alternaria.  No  such 
coloration  as  this  has  been  observed  in  my  cultures.  The  same  author  (91) 
discussing  colony  zonation,  attributes  it  to  variation  in  the  color  of  the  my- 
celium, while  in  my  cultures  it  appears  to  be  almost  entirely  due  to  variation 
in  quantity  of  conidia  and  of  aerial  mycelium. 

The  quantity  of  aerial  mycelium  was  highly  dependent  upon  air- 
moisture  and  agar-conditions.  When  these  favored  there  was  a  some- 
what conspicuous,  even  floccose,  white  aerial  mycelium  2  to  3  mm.  high, 
consisting  of  either  smooth,  even,  single  filaments  or,  when  quite  abun- 
dant, of  several  filaments  twined  into  a  rope  (Fig.  5,  c] .  When  conditions 
were  less  favorable  the  aerial  mycelium,  though  inconspicuous,  was  still 
present  in  considerable  quantity.  The  difference  between  various  species 
of  Helminthosporium  and  their  saltants  as  regards  the  abundance  of 
aerial  mycelium  is  well  shown  in  Plates  IX-XIII.  H.  No.  36,  a  very  dis- 
tinct species  from  H.  No.  1,  was  chiefly  characterized  by  the  great 
profusion  of  aerial  mycelium  (PI.  X).  This  character  is  the  only  one 
I  have  found  by  which  to  separate  H.  No.  1  and  H.  No.  3,  and  it  serves 
only  in  the  large  cultures  (PI.  IX-XIII),  the  distinction  often  failing  in 
ordinary  Fetri-dish  culture.  The  character  is  apparently  so  much  mod- 
ified by  varying  humidity  as  to  make  its  utility  for  diagnosis  of  very 
questionable  value  (see  pp.  95,  97).  Ravn  (91),  however,  distinguishes  H. 
avenae,  H.  gramineum,  and  H.  teres,  growing  on  sterilized  straw,  by  the 
different  individual  characters  of  the  aerial  mycelium  and  sclerotia;  and 
H.  gramineum  and  H.  avenae  on  beerwort  by  the  fact  that  PI.  gramineum 
forms  a  snow-white  uniform  mass  of  aerial  hyphae,  while  H.  avenae  has 
sparse,  white  aerial  mycelium  which  forms  solid  clumps,  the  black  sub- 
stratum-mycelium being  visible  between  them. 

The  mycelium  in  wheat  tissue  is  somewhat  more  irregular  in  contour 
than  that  grown  in  agar  and  is  often  much  thicker.  Occasionally  upon  the 
wheat  surface  it  branched  in  a  close  fan-like  fashion  (Fig.  22,  page  134) .  The 


106 


FIG.  6. — H.  No.  1:  d,  showing  branching  and  general  appearance  at  edge  of  a 
colony  growing  on  corn-meal  agar;  e,  portion  of  a  mycelial  clump  showing 
peculiar  twisted  character  of  the  mycelium,  with  more  usual  strands  for  compari- 
ison. 


107 

aerial  mycelium  of  some  races  produced  clumps  (PI.  XIII,  XXII,  XXIII), 
which  under  the  microscope  are  seen  to  be  due  to  a  peculiar  distortion  and 
abundance  of  the  aerial  mycelial  tips  (Fig.  6,  d).  This  peculiar  behavior 
of  the  terminal  parts  of  the  mycelium  shows  some  similarity  to  the  branch- 
ing figured  by  Ward  (123)  in  Botrytis  in  the  early  stages  of  development  of 
attachment  organs.  Anastomosis  is  very  common  with  this  fungus  (Fig. 
5,  a  and  b),  and  Ward  (123,  fig.  19)  has  figured  anastomosis  of  very  similar 
character  for  Botrytis.  Many  citations  of  its  occurrence  are  given  by 
Beauverie  and  Guilliermond  (13).  (See  also  their  figures  4  and  8.) 

The  nuclei  in  the  mycelium  are  extremely  small,  but  may  be  seen  readily 
when  stained  with  gentian  violet,  and  still  better  if  stained  with  iron-hae- 
matoxylin.  They  vary  in  number,  but  are  never  less  than  two  and  usually 
more;  they  do  not  typically  group  in  pairs;  and  they  are  irregularly  dis- 
tributed in  the  protoplast.  Nuclei  apparently  in  mitosis  are  frequently 
seen,  but  since  they  are  so  small  no  details  were  noted  except  that  the 
mitoses  of  all  of  the  nuclei  in  one  cell  seem  to  be  simultaneous,  and  mitosis 
was  probable  in  adjacent  cells.  Reports  on  the  nuclear  conditions  in  the 
fungi  imperfecti  are  few  and  unsatisfactory,  doubtless  owing  to  the  extreme 
difficulty  of  the  subject.  Dangeard  (39)  notes  nuclei  and  mitosis  in  the 
rather  anomalous  genus  Bactridium;  Beauverie  and  Guilliermond  (13)  in 
Botrytis;  while  Higgins  (66)  gives  quite  satisfactory  figures  for  Mycosphae- 
rella. 

Senescence  phenomena  of  aerial  mycelium  (Fig.  7,  a — b). —  When  the 
aerial  mycelium  is  young  it  constitutes  a  more  or  less  abundant,  loose, 
arachnoid,  fluffy  mass.  In  quite  old  cultures  it  is  observed  to  mat  down 
close  to  the  surface  of  the  medium  in  a  thin,  glazed,  dead  layer.  Inter- 
mediate between  these  two  extreme  conditions  interesting  phenomena 
occur.  The  first  observable  change  from  that  of  the  normal,  vigorous 
mycelium  is  that  certain  cells  of  a  filament,  often  many  adjacent  cells,  be- 
come nearly  or  quite  devoid  of  protoplasm  (Fig.  7,  i,  o,  and  g] ,  though  cells 
at  each  end  of  such  a  series  still  appear  normal  (see  i  and  j).  Quite  fre- 
quently the  fungus  re-grows  from  a  protoplasmic  cell,  through  the  empty 
threads,  as  is  shown  in  n  and  o.  In  other  instances,  and  much  more  com- 
monly, the  empty  cells  gradually  collapse  until  they  remain  as  very 
thin,  smooth  filaments  (c,  h,  and  m),  apparently  of  gelatinous  texture. 
Where  two  filaments  undergoing  such  dissolution  cross  they  blend  (a,  b, 
m) ;  and  where  several  meet,  rather  large  amorphous  unorganized  masses 
are  seen,  superficially  much  resembling  a  plasmodial  structure  (a).  There 
was,  indeed,  at  first,  suspicion  that  there  might  be  present  a  plasmodial 


108 

parasite  preying  upon  the  Helminthosporium  mycelium;  but  numerous 
tests  convinced  me  that  such  was  not  the  case,  but  that  what  really  occurs 
is  that  the  old  aerial  mycelium  dissolves  (probably  by  auto-digestion).  All 
stages  of  this  disorganization  can  be  followed  under  the  immersion  leris  in 
stained  preparations,  where  the  disorganized  filament  stains  wifn  the  gen- 
tian violet  but  is  seen  to  be  amorphous  and  without  protoplasmic  content. 
These  phenomena  appear  to  be  limited  to  the  aerial  mycelium,  but  were 


FIG.  7. — Various  views  (a  and  b,  low  power,  c — o,  high  power)  of  mycelium  of  H.  No.  1  in 
senescence:  o  and  b  showing  dissolution  to  fine  threads,  b  with  a  conidipphore  still  attached; 
c — h  and  m,  empty  mycelial  cells  adjacent  to  cells  nearly  dissolved;  i  and  j,  protoplasmic 
cells  adjacent  to  empty  cells;  k  and  I,  fine  mycelial  outgrowths  from  protoplasmic  cells;  «  and 
o,  fine  mycelia-l  threads  growing  from  the  protoplasmic  cells  and  through  old  empty  cells;  p, 
bits  of  mycelium,  as  seen  with  the  immersion  lens,  showing  the  nuclei. 

observed  on  many  strains  of  Helminthosporium.  Autodigestion  of  my- 
celium doubtless  occurs  in  the  case  of  wood-rotting  fungi,  as  is  evidenced 
by  the  absence  of  mycelium  where  it  was  previously  known  to  be,  and  it 
may  be  of  common  occurrence  in  other  fungi.  It  certainly  occurs  when 
two  hyphae  join  by  anastomosis,  and  in  the  union  of  sexual  organs.  Grow- 
ing-through of  the  mycelium,  as  noted  above,  and  even  conidia-formation 
within  the  old  cell  are  common  in  Saprolegnia,  and  have  been  noted  in 


109 


several  other  genera:  Botrytis  (Beauverie  and  Guilliermond,  13);  Alter- 
naria,  Epicoccum,  and  Botrytis  (Linder,  78);  Inzengaea  (Borzi,  23);  De- 
matium,  Botrytis,  Oidium  (Klocker  and  Schiorming,  75);  Chaetomium 
(Zopf,  129,  figs.  24,  25,  A,  B,  Tab.  16).  The  phenomenon,  as  described, 
is  always  associated  with  senescence.  Sclerotia  are  described  by  Bakke 
(6)  and  by  Noack  (87) ,  who  seem  to  have  found  them  common  on  old  straw- 
cultures,  varying  in  length  from  200  to  600  /*.  I  have  not  found  them  at  all 
on  straw,  though  on  old  rice-cultures  they  are  abundant.  Pycnidia  and 
pycnoconidia,  as  seen  by  Ravn  (91)  in  H.  teres  and  as  described  by  Bakke 
(6),  I  have  not  seen. 

CONIDIOPHORES 

On  standard  wheat- shoots. — The  conidiophores  are  in  no  sense  clustered 
but  arise  singly  as  lateral  branches,  each  from  an  ordinary  mycelial  cell, 
and  differ  from  the  mycelium  chiefly  in  that  they  grow  erect  and  straight 
instead  of  declined  and  crooked,  and  are  darker  in  color  than  the  mycelium. 
Usually  this  branch  in  its  basal  region  is  mycelium-like,  but  it  rapidly 
thickens  and  darkens  to  true  conidiophore  character,  and  is  usually  2.5  to 
5  n  in  length.  Sometimes  the  mycelial  cell  from  which  the  conidiophore 
arises  also  darkens.  The  conidiophore-cells  contain  protoplasm,  and  the 
protoplast  plasmolizes  under  the  usual  reagents.  When  mature  the  conid- 


FIG.  8.  —  H.  No.  1,  showing  variation  in  conidiophores,  geniculation, 
conidia-scars,  and  septation. 


110 

iophores  are  pale  straw-color  to  smoky  brown  from  tip  to,  or  nearly  to, 
the  base,  the  color  being  due  to  the  outer  wall — which  is  very  brittle.  Upon 
the  production  of  the  first  conidium,  which  is  strictly  terminal,  the  conid- 
iophore  grows  onward,  with  a  slight  bend  where  the  first  conidium  was 
produced,  and  proceeds  to  bear  another  one.  This  may  contTnue  until 
many  conidia  have  been  borne  by  the  same  conidiophore.  If  the  conidia 
are  undisturbed,  the  cluster  may  have  a  botryose  effect,  but  if  disturbed, 
only  the  youngest  conidia  remain,  and  scars  and  geniculations  mark  the 
places  of  origin  of  the  fallen  conidia  (Fig.  8) .  The  number  of  conidia  borne 
per  conidiophore  in  count  of  24  was  as  follows: 

Frequency,  15,  5,  4 
Conidia,         1,  2,  3 

Much  higher  numbers  than  this  occasionally  occurred  under  standard  con- 
ditions (see  appendix,  page  180) ;  and  much  higher  numbers  were  the  rule 
on  corn-meal  agar. 

The  number  of  septa  below  the  first  scar  varied  from  one  to  four, 
while  the  length  in  seven  measurements  from  base  to  first  scar  was  78 — 
88  /it.  The  length  above  the  first  scar  is  entirely  dependent  upon  the  num- 
ber of  conidia  borne  on  a  given  conidiophore;  in  some  cases  it  is  equal  to 
or  even  greater  than  the  length  below  the  first  scar. 

Conidia  develop  very  rapidly  upon  the  conidiophores.  One  of  the 
latter  kept  constantly  under  observation  was  first  observed  at  11  o'clock 
to  have  a  diameter  of  6.8  /A;  at  11 :15,  13.6  p;  at  11 :30,  20.4  ju;  at  12,  37.4  /*; 
and  at  12:30,  44.2  M. 

The  conidiophores  of  certain  other  numbers,  for  example  H.  Nos.  2, 
21,  and  29,  are  of  such  very  different  character  that  the  conidiophores  alone 
serve  to  distinguish  them  markedly  from  H.  No.  1.  The  conidiophores  of 
H.  Nos.  3,  5,  15,  16,  and  others,  however,  are  closely  like  those  of  H.  No.  1; 
indeed  no  real  distinction  could  be  found  between  them.  Attempts  were 
made  to  distinguish  between  these  strains  or  species  by  plotting  conidio- 
phore length,  septation,  length  of  cells,  etc.,  but  nothing  came  of  such  at- 
tempts. The  length  of  the  conidiophore  is  markedly  influenced  by  air- 
humidity  (page  95),  and  it  is  probable  that  the  rudimentary  conidiophores 
may  be  changed  into  aerial  mycelium  by  a  lowering  of  the  air-humidity. 

CONIDIA 

The  conidia  and  their  attachment  to  the  conidiophores  are  shown  in 
Plate  XVII.  From  the  basal  end  of  the  conidium  to  the  conidiophore 
there  is  an  exceedingly  short  (2X4  /z)  black  stipe.  As  the  conidium  falls 


Ill 

away  from  the  conidiophore  the  stipe  remains  attached  to  the  conidium, 
and  as  it  can  always  be  seen  readily  when  the  conidium  is  in  suitable  posi- 
tion, it  serves  as  a  ready  means  of  recognizing  the  basal  end  of  the  conidium. 
The  stipe  is  equally  obvious  and  distinguishable  in  H.  Nos.  3,  4,  5,  13-16, 
etc.,  though  in  H.  No.  2  and  certain  other  numbers  the  stipe  is  of  somewhat 
different  type.  While  in  very  rare  instances  catenulation  of  conidia  was 
observed  (Fig.  9,  b] ,  this  is  apparently  much  less  frequent  than  in  the  forms 


FIG.  9.— H.  No.  1:  a,  portion  of  a  conidiophore  bearing 
conidia;  b,  catenulate  conidia — rarely  occurring. 

described  by  Ravn.  The  apical  end  of  the  conidium  is  always  obtuse,  and  is 
marked  by  a  pale  spot  that  was  mentioned  by  Ravn  (91)  as  occurring  in 
H.  teres,  etc.  Being  of  so  distinctive  a  character,  this  end  is  always  recog- 
nizable when  the  conidium  is  in  a  suitable  position.  We  have,  then,  reliable 
means  of  identifying  each  end  of  the  conidia:  the  basal  stipe  and  the  apical 
spot  (Fig.  10).  The  latter,  though  not  characteristic  of  all  Helmintho- 
sporiums — for  example,  H.  Nos.  2,  29,  and  others  lack  it — is  characteristic 
of  H.  Nos.  1,  3,  13-16,  and  others. 

The  color  of  the  conidia  of  H.  No.  1  ranges  from  pale-straw  to  light 
brown,  and  under  some  conditions  shows  a  slight  bluish-green  tinge.  While 
H.  Nos.  2  and  28  were  distinctly  and  constantly  different  from  H.  No.  1  in 
color,  H.  Nos.  1,3,  13-16,  etc.,  were  indistinguishable  on  a  color  basis. 

The  conidial  outer  wall. — This  wall  (the  episporium  of  de  Bary,  9), 
which  gives  the  color  to  the  conidium,  is  extremely  thin  and  very  fragile 


112 


(PL  XVIII).  It  is  so  brittle  that  by  gently  tapping  the  cover-glass  over 
conidia  the  outer  dark  wall  of  every  one  of  them  may  be  broken  in  frag- 
ments, much  as  a  peanut  is  broken  if  stepped  upon.  This  character 
is  common  to  H.  Nos.  1,  3,  5,  13-16,  etc.,  as  well  as  to  H.  HO.  2, _and  many 
other  species,  though  in  some  the  wall  is  less  brittle  than  in  others.  The 
conidial  wall  that  is  left  after  the  solution  of  the  endosporium  by  sulfuric 
acid  is  entirely  without  sign  of  septation,  but  shows  the  apical  spot  clearly 
differentiated  as  a  thin  pale  region. 


FIG.  10. — Variation  in  conidial  shape  and  septa- 
tion of  H.  No.  1,  and  showing  also  the  dark  spot, 
stipe,  at  basal  end,  and  the  pale  apical  spot. 

Conidial  contents. — Within  the  thin  colored  wall  are  the  protoplasts, 
usually  vseveral  in  number  (Fig.  11),  and  between  the  protoplasts  and  the 
outer  wall  is  a  thick  hyaline  layer  of  substance  that  is  somewhat  soft,  usually 
appearing  almost  gelatinous  (PL  XVIII).  This  hyaline  soft  layer  represents 
morphologically,  I  believe,  a  second  cell-wall,  the  endosporium  of  de  Bary 
(9).  I  shall  so  speak  of  it.  That  this  wall  is  soft  is  shown  by  the  way  the 
conidial  contents  issue  from  the  end  of  a  cracked  conidium  under  pressure 


113 


FIG.  11.— Conidia  of  H.  No.  1:  a  and  b, 
with  outer  wall  cracked  open  by  pres- 
sure, the  inner  hyaline  wall  and  the  pro- 
toplasts emerging;  c,  another  conidium  with 
the  outer  wall  crushed  by  pressure,  the  two 
protoplasts  walled  and  touching;  d,  similar 
to  c,  but  with  the  protoplasts  separate;  e, 
immersion-lens  view  of  two  protoplasts 
within  a  conidium,  showing  thickening  at 
their  point  of  nearest  approach  to  each 
other;  /,  a  longitudinal  microtome-section 
of  a  conidium  from  which  both  sides  have 
been  cut  away;  g,  a  cross-section  of  a 
conidium  showing  much  clear  space  between 
the  protoplast  and  the  outer  wall. 

(Fig.  11).  De  Bary  (9)  remarks  that  the  endosporium  often  shows  great 
softness  and  delicacy  but  is  by  no  means  always  thinner  than  the  other 
wall.  That  the  outer  conidial  wall  has  no  internal  ridges,  and  takes  no 
part  in  forming  septa,  is  shown  both  by  direct  observation  and  by  inference 
from  the  way  in  which  the  conidial  contents  slide,  unobstructed,  length- 
wise of,  and  out  of,  the  outer  conidial  wall.  Ravn  (91)  states  that  in  the 
three  species  studied  by  him  the  walls  and  septa  are  very  thin,  but  when 
treated  with  glycerine,  etc.,  the  outer  wall  becomes  prominently  thickened, 


114 

as  also  the  cross  walls  where  they  meet  the  outer  wall.  In  making  this 
statement  he  refers  to  the  episporium  and  endosporium  as  constituting  two 
layers  of  one  wall.  In  some  instances  the  endosporium  is  clearly  seen  to 
extend  between,  and  to  separate,  the  protoplasts  (Fig.  11,  &Xi  while  in  other 
cases  the  protoplasts  appear  to  touch  each  other  ( Fig.  11,  a) ,  yet  when  the 
conidial  contents  are  pushed  from  a  crushed  conidium  there  is  always  a 
line,  though  sometimes  it  is  very  thin,  separating  the  protoplasts.  Since 
the  protoplasts  are  distinct  from  each  other,  and  are  thus  separated  by  the 
endosporium,  it  seems  justifiable  to  assume  that  this  second  cell-wall  forms 
the  septa,  sometimes  obvious  though  very  thin,  between  the  protoplasts. 
Treated  with  concentrated  sulfuric  acid  the  conidial  endosporium  dissolves 
rapidly,  and  by  the  generated  pressure  the  episporium  is  ruptured,  invari- 
ably at  the  basal  end  first,  this  often  opening  trap-door-like,  though  fre- 
quently the  pressure  is  sufficient  to  tear  the  wall  of  the  conidium  open 
throughout  its  length.  With  the  solution  of  the  endosporium  the  proto- 
plasts issue  from  the  case  of  the  conidium  and  appear  to  be  unattached. 

The  individual  protoplasts  vary  much  in  shape,  sometimes  being  nearly 
spherical;  in  other  cases  nearly  cubical.  Each  protoplast  is  surrounded 
by  a  differentiated  layer  which  in  some  cases  is  so  clear,  distinct,  and  thick 
as  to  appear  to  be  a  third  wall  (Fig.  11).  Perhaps  it  is.  Under  gentian 
violet  and  many  other  aniline  stains,  while  the  protoplast  takes  a  strong 
stain  this  layer  refuses  to  stain.  Microtome  cross-sections  and  longitudi- 
nal sections  of  conidia  verify  the  foregoing  conclusions  (Fig.  11,  /).  In 
cross-sections  (Fig.  11,  g),  with  Fleming's  triple  stain  the  protoplast  stains 
as  usual,  but  the  second  cell-wall  refuses  to  stain;  under  Bismarck  brown  it 
takes  a  very  faint  stain.  Under  action  of  aniline  blue,  iodine,  fuchsin,  mal- 
achite green,  Pianese,  or  chlor-zinc-iodine  it  remains  unstained.  In  longi- 
tudinal sections  cut  so  thin  that  two  sides  of  the  conidium  have  been  cut 
away,  mature  conidia  show  no  continuity  of  the  protoplasts  (Fig.  11,  d). 
When  plasmolized  the  protoplasts  all  shrink  and  lie  quite  separate  from  each 
other,  and  it  is  in  such  condition  that  the  appearance  of  a  third  conidial 
wall  is  most  evident  (Fig.  11,  a,  b) .  Previous  to  plasmolysis  the  proto- 
plasts are  frequently  seen  to  touch  each  other  on  the  median  longitudinal 
axis  of  the  conidium,  and  a  very  faint  line  (plane)  is  observable  extending 
across  the  conidium  (Fig.  11,  a,  b).  This  probably  represents  the  true 
septum,  following  nuclear  division.  The  protoplast  wall  bears  a  small 
dot-like  thickening  (Fig.  11,  e)  adjacent  to  its  sister  protoplast,  which  may 
also  be  residual  evidence  of  nuclear  mitosis. 

The  characters  as  here  given  for  H.  No.  1  are  found  also  in  such  re- 


115 


lated  forms  as  H.  Nos.  3,  13,  16,  etc.  That  the  internal  structure  of  Hel- 
minthosporium  conidia  has  not  been  clearly  understood  is  shown  by  nu- 
merous published  figures. 

Conidial  germination. — The  conidia  germinate   readily   in   water,   in 
hanging  drop,  or  on  the  surface  of  wheat  shoots  (Fig.  12),  and  germination, 


FIG.  12. — Germinating  conidia  of  H.  No.  1 

so  far  as  I  have  seen,  is  very  rarely  lateral  but  usually  from  the  ends,  most 
commonly  from  the  basal  end.  Thus  twenty-seven  basal  germinations 
were  counted  as  against  fourteen  apical  ones.  The  germ-tube  is  hyaline, 
richly  filled  with  protoplasm,  and  forms  abundant  branches  and  septa 
(Fig.  12).  Bakke  (6)  states  that  "germ  tubes  first  come  from  basal  and 
apical  cells;  later  other  germ  tubes  may  arise  from  the  remaining  cells  under 
favorable  conditions."  Kirchner  (72)  states  that  germination  in  H.  gramin- 
eum  is  usually  terminal,  but  Noack  (87)  shows  that  for  this  species  the 
germ-tubes  are  as  often  lateral.  The  viability  of  the  protoplast  was  not 
injured  by  crushing  the  epispore;  indeed  such  cracking  seemed  to  facilitate 
emergence  of  the  germ-tube.  Anastomosis  of  the  germ-tubes  is  not  uncom- 
mon (Fig.  13).  As  the  germ-tube  enlarges  there  is  frequently,  though  not 


FIG.  13.— Conidiaof  H.  No.  1: 
a,  showing  septa  from  two 
depths  of  focus;  b,  two  germinat- 
ing conidia  with  germ-tubes 
anastomosing. 


always,  shrinkage  of  the  protoplasts  such  as  is  shown  in  Fig.  1 1,  this  shrink- 
age being  usually  most  pronounced  in  the  end  of  the  conidium  showing  most 
vigorous  growth.  To  all  appearance  the  endosporium  serves  as  a  stored 
food  and  is  consumed  in  germination,  since  its  presence  in  much  diminished 
quantity  in  germinated  conidia  is  evident  when  such  conidia  are  crushed. 
The  conidiophore-cells  also  occasionally  function  as  conidia  by  sending 
out  a  germ-tube.  Here,  too,  the  inner  cell-wall  seems  to  serve  as  reserve 
food. 

Longevity  of  conidia. — It  is  not  known  how  long  conidia  live,  but  on 
wheat  straw  that  had  remained  air-dry  for  fourteen  months  they  germi- 
nated normally.  Noack  (87)  mentions  germination  "after  many  months." 
Ravn  (91)  says  of  three  species  that  at  eight  months  they  germinated  but 
sparingly  or  not  at  all. 

Frequency  of  conidial  septa. — H.  No.  1  under  standard  conditions  (see 
appendix,  page  180)  gave  the  graph  in  Fig.  J,  while  similar  data  for  H.  No?. 
13-16  are  given  in  Fig.  T. 


117 


Septa  Differences  of  means  of  septa 

H.  Nos.  -  1  and    3 0.27  ±  .16 

H.  Nos.    1  and  15 0.80  =*=  .12 

H.  Nos.    land  16... 0.58  ±.11 

H.  Nos.  15  and  16 0.22  =±=  .11 

It  is  to  be  noted  that  the  differences  between  Nos.  15  and  16  are  quite 
as  large  relative  to  the  probable  error  as  are  the  differences  between  Nos.  1 
and  3.  Ravn  (91) ,  speaking  of  three  species  of  Helminthosporium,  says  that 
the  septa  are  very  variable,  and  that  specific  differences  can  not  be  derived 
from  them.  Very  abnormal  septation  was  frequent;  for  example,  on  green- 
wheat  agar  (Fig.  14)  and  other  uncongenial  media. 


FIG.   14. — Various  abnormal  conidia  of 
H.  No.   1  as  grown  on  green-wheat  agar. 

Conidial  shape. — The  shape  of  the  conidia,  together  with  their  size  and 
septation,  are  in  the  genus  Helminthosporium  the  three  most-used  charac- 
ters in  description.  Indeed  in  published  descriptions  of  many  species  these 
are  the  only  important  characters  mentioned,  and  often  one  or  more  of  these 
is  lacking.  Conidial  shape  in  certain  species  is  very  characteristic,  partic- 
ularly in  H.  inaequalis,  H.  geniculattim,  H.  ravenelii,  and  also  in  my  H. 
No.  29.  Much  stress  has  been  laid  on  conidial  shape  as  a  means  of  distin- 
guishing certain  cereal  Helminthosporiums,  particularly  in  distinguishing 
H.  sativum  from  //.  teres. 

Merely  to  look  at  two  lots  of  conidia  with  the  microscope,  even  with 
the  aid  of  a  comparison  ocular,  is  not  a  satisfactory  means  of  ascertaining 
the  prevailing  conidial  shape.  Many  strains  of  Helminthosporium  vary 
greatly  as  to  conidial  shape,  and  conidia  of  one  shape  are  mixed  with  those 
of  another  (PI.  XIX— XXI).  The  important  question  is,  what  is  the 
relative  frequency  of  the  various  shapes?  But  before  any  fair  estimate  of 
this  can  be  made,  standards  must  be  established  as  to  what  are  the  essen- 
tial characters  of  the  various  shapes. 

One  factor  of  preponderating  influence  in  determining  these  conidial 
shapes  is  the  position  that  the  point  of  greatest  diameter  occupies  on  the 


118 


FIG.  15. — Diagrams  elucidating  conidial  shape. 


longitudinal  axis  of  the  conidium.  In  diagrams  I  and  II,  Fig.  15,  the 
point  of  greatest  diameter  on  the  line  a — a'  is  midway  between  the  base 
and  apex  of  the  conidium;  while  in  diagrams  III  and  IV  it  is  nearer  to  its 
base.  If  the  conidium  tapers  from  the  point  of  greatest  thickness  toward 
each  end  a  fusiform  (Diagr.  Ill)  or  elliptical  (Diagr.  I,  1,  2)  conidium  re- 
sults. If  for  a  sufficient  distance  on  each  side  of  the  line  a — a'  the  conidium 
remains  of  uniform  diameter  it  approaches  more  nearly  the  form  of  a  cyl- 
inder (Diagr.  II,  1,  2).  When  the  maximum  diameter  is  nearer  to  the  base 
than  to  the  apex,  somewhat  rapid  tapering  gives  a  fusiform  conidium  (Diagr. 
Ill) ,  but  if  (as  in  Diagr.  IV)  the  diameter  lessens  very  gradually,  as  from  the 
point  a  to  point  y,  the  conidium  may  be  said  to  be  subcylindrical. 


119 

Mere  casual  observation  of  many  strains  of  Helminthosporium  showed 
that  the  point  of  maximum  diameter  was  usually  near  the  basal  region  of 
the  conidium,  occasionally  near  the  middle  region,  while  in  extremely  rare 
cases  it  was  near  the  apical  quarter,  the  ratio  of  these  cases  being  for  H. 
No.  1  about  30:14:4.  A  more  accurate  determination  of  what  may  be 
termed  the  longitudinal  eccentricity  of  the  conidium — that  is  the  range 
of  variation  in  the  position  of  the  line  of  greatest  diameter  (a — a ' ,  Diagr. 
I — IV)  may  be  made  by  measuring  (along  the  longitudinal  axis)  the  dis- 
tance from  the  base  of  the  conidium  to  the  intersection  of  the  line  a — a! 
with  the  longitudinal  axis.  This  distance  divided  by  the  total  length  of 
the  conidium  may  be  known  as  its  coefficient  of  longitudinal  eccentricity. 
This  coefficient  for  H.  No.  1,  based  on  65  conidia  taken  at  random,  was 
found  by  the  above  method  to  be  .43  ±0.  In  other  terms  the  point  of 
maximum  diameter  was  distant  from  the  base  of  the  conidium  43%  of  the 
total  length  of  the  conidium.  Bakke  (6)  says  that  the  conidia  of  H.  te- 
res  are  widest  at  the  middle.  The  coefficient  of  longitudinal  eccentricity 
based  on  11  conidia  of  H.  No.  1  which  were  of  typical  subcylindrical  ap- 
pearance (approaching  that  shown  in  Diagr.  IV)  was  .45  as  contrasted  with 
a  coefficient  of  .43  for  11  conidia  of  elliptical  appearance  (Diagr.  I).  Co- 
efficients of  longitudinal  eccentricity  for  H.  Nos.  5,  20,  and  4  of  subcylin- 
drical shape,  were  respectively  .35,  .39,  and  .37,  showing  that  in  these 
forms  the  point  of  maximum  diameter  is  slightly  nearer  the  base  than  it  is 
in  H.  No.  1.  None  of  the  conidia  of  H.  No.  1  was  truly  cylindrical, 
that  is,  the  sides  were  not  parallel  for  any  appreciable  distance.  Many 
were  subcylindrical,  the  form  approaching  that  shown  in  Diagram  IV.  Of 
65  conidia  taken  at  random  81%+  of  the  conidia  were  elliptical;  17%  + 
subcylindrical;  and  1%  otherwise. 

To  secure  a  coefficient  which  would  indicate  with  some  degree  of  ac- 
curacy the  curvature  of  the  conidial  wall  (as  from  point  a  to  point  y,  Diagr. 

xy 
I — IV)  determinations  were  made  of  the  ratio  —  •  (Diagr.  I — IV).      The 

£> 

line  cd  was  tangential  to  the  surface  of  the  conidium  at  the  point  of  maxi- 
mum diameter,  and  was  parallel  to  the  longitudinal  axis  of  the  conidium, 
the  line  ef  being  3.4  /*  from  the  line  cd  and  parallel  to  it.  Then  the  points 
x  and  y  are  where  the  surface-line  of  the  conidium  cuts  the  line  ef.  It  is 
obvious  that  as  the  line  xy  increases  in  proportion  to  the  length  of  the  conid- 
ium, gh,  the  conidium  more  nearly  approaches  the  form  of  a  cylinder;  and 
as  the  line  xy  becomes  proportionately  shorter  the  conidium  becomes  less 

xy 
like  a  cylinder.     The  ratio   —   may    therefore   be   termed    the   coefficient 


120 

of  cylindricity.  For  these  determinations  only  conidia  of  approximately 
modal  length  were  used,  and  to  obviate  unconscious  selection,  measure- 
ments were  made  of  only  the  left  side  of  the  conidium,  the  basal  end  being 
toward  the  observer.  Determination  from  11  conidia  of  H.  No.  1  of  sub- 
cylindrical  shape  gave  a  coefficient  of  .74,  while  that  frorrPST  elliptical 
conidia  was  .67. 

The  above  findings  for  H.  No.*l  are  as  follows: 

Coefficient  of  longitudinal  eccentricity 

All  conidia 43 

Elliptical  conidia 42 

Subcylindrical  conidia 45 

Coefficient  of  cylindricity 

All  conidia 70 

Elliptical  conidia 67 

Subcylindrical  conidia 74 

Determinations  of  the  coefficient  of  cylindricity  made  from  drawings 
of  Dr.  Ravn  (91)  gave  for  H.  gramineum  and  H.  avenae  respectively  .86  and 
.95,  showing  a  much  higher  coefficient  than  is  given  by  any  of  the  forms  in 
my  collection. 

A  convenient  method  of  measuring  conidia  for  coefficients  is  given  on 
page  179  of  the  appendix. 

Conidial  length. — From  five  separate  plates,  a,  b,  c,  d,  and  e,  inoculated 
with  H.  No.  1  under  standard  conditions,  Graphs  36-40  (Fig.  K)  of  conidial 
length  were  made.  Two  additional  graphs  were  made  from  plate  e,  one 
of  which  is  designated  as  e1 '.  The  data  pertaining  to  these  graphs  are 
given  with  the  others  (Fig.  K). 

The  differences  between  the  means  of  conidial  length  on  plates  a  to  e 
and  e'  are  as  follows: 

Plates       Differences  between  means  Plates       Differences  between  means 


a-b  +0.70  ±  .21 

a-c  +0.62  ±  .24 

a-d  -0.78  ±  .23 

a-e  +2.03  ±  .24 

a-e'  +0.50  ±  .16 

b-c  -0.07  ±  .22 

b-d  +0.08  ±.23 


b-e'  -0.19  ±  .16 

c-d  +0.16  ±  .23 

c-e  +  1.40  =»=  .24 

c-e'  +0.11  ±  .17 

d-e  +1.24  ±.25 

d-e'  -0.28  ±.18 

e-e'  -1.52  ±  .19 


b-e  +1.32  ±  .24 

Since  the  various  plantings  on  these  plates  were  all  from  the  same  in- 
oculum, made  at  the  same  time,  and  under  as  nearly  identical  conditions 
as  possible,  and  so  kept,  the  rather  large  difference  in  means  seen,  particu- 


121 


larly  in  a — e,  b — e,  c — e,  d — e,  and  e — e'  is  significant.  If  plate  e  be  left 
out  of  consideration,  the  others  agree  reasonably  well,  with  differences 
greater  than  the  probable  error  in  six  out  of  ten  cases,  the  difference  being 
but  slightly  above  the  probable  error  in  two  cases,  about  twice  the  probable 
error  in  two  cases;  and  about  thrice  that,  in  two  cases,  the  largest  excess, 
in  plates  a — b,  being  0. 70 ±.21. 

Plate  e  deviates  widely,  with  a  difference  in  case  of  a — e  of  2.03  ±24, 
the  difference  being  more  than  eight  times  the  probable  error.  The  great 
difference  in  plate  e  must  indicate  variability  of  the  fungus  on  this  plate 
(cf.with  page  152), modification  due  to  influence  of  some  unknown  factor  of 
environment,  or  error  in  sampling.  But  since  such  a  variation  did  occur 
in  a  series  of  plates  made  with  the  greatest  care  and  with  the  same  organ- 
ism, it  is  clear  that  the  occurrence  of  such  a  difference  can  not  properly  be 
interpreted  as  meaning  specific  difference.  Data  from  the  combined  rec- 
ords of  a,  b,  c,  d,  and  e'  (omitting  e  as  questionable)  give  the  most  reliable 
data  I  have  on  length  of  conidia  of  H.  No.  1  under  standard  conditions 
(cf.  with  Graph  42,  Fig.  K). 

To  determine  how  wide  a  variability  occurs  in  specimens  collected  in  the 
open,  on  the  natural  host,  H.  ravenelii  (PI.  XX)  a  well-marked,  easily  recog- 
nized species  of  wide  geographic  distribution,  growing  on  Sporobolus,  was  stud- 
ied in  conidial-length  graphs  made  from  specimens  listed  in  connection  with 
the  graphs  (Fig.  L) .  The  tabulated  results  of  this  study  of  H.  ravenelii  follow : 


Nos* 
43—46 
43—47 
43—49 
43—48 
43—50 
43—51 
43—52 
43—53 
44-^8 
44—49 
44—50 
44—51 
44—52 
44—53 
45—50 
45—51 
45—52 
45—53 


Differences  between  means 


Nos.*     Differences  between  means 


0.48 
0.59 
0.62 
0.62 
0.98 
1.18 
1.94 
2.92 
0.45 
0.46 
0.81 
1.01 
1.77 
2.75 
0.80 
1.00 
1.76 
2.74 


.26 

.24 
.28 
.29 
.27 
.27 
.27 
.26 
.31 
.31 
.30 
.30 
.30 
.29 
.24 
.25 
.25 
.24 


46—50 
46—51 
46—52 
46—53 
47—51 
47—52 
47—53 
48—51 
48—52 
48—53 
49—51 
49—52 
49—53 
50—52 
51—52 
51—53 
52—53 


0.50 
0.70 
1.46 
2.43 
0.59 
1.35 
2.33 
0.56 
1.32 
2.30 
0.55 
1.31 
2.29 
0.96 


.28 
.28 
.28 
.27 
.26 
.26 
.25 
.30 
.30 
.29 
.29 
.29 
.29 
.29 


0.76  =t  .29 
1.73  ±  .28 
0.97  ±  .28 


*For  significance  of  numbers,  see  Figure  L. 


122 

A  difference  of  0.97^.28  between  the  means  of  two  samples  from  the 
same  specimen  (Nos.  52  and  53) — a  difference  more  than  three  times  the 
probable  error — shows  clearly  the  difficulties  of  sampling,  and  that  such 
differences  between  samples  of  the  same  species  grown  under  the  same  con- 
ditions may  be  expected.  The  differences  in  several  instances,  notably 
between  Nos.  44  and  49,  44  and  48,  and  46  and  51,  are  no  greater  than  those 
between  two  samples  of  the  same  specimen  and  may  well  be  due  to  sampling, 
and  to  this  extent  show  the  fungus,  over  a  wide  geographic  range,  to  be 
remarkably  uniform.  In  several  other  instances,  however,  there  is  a  wide 
difference  of  means,  above  the  probable  error — notably  in  all  cases  involv- 
ing sample  No.  53.  These  differences  are  often  four,  five,  or  six  times  the 
probable  error,  and  occasionally  run  as  high  as  eleven  or  twelve  times  the 
probable  error  even  with  this  remarkably  uniform  fungus.  While  these 
differences  may  in  part  be  attributed  to  sampling  they  probably  represent 
also  morphological  changes  due  to  environmental  differences,  and  differ- 
ences of  nutrition  or  humidity,  but  do  not  necessarily  indicate  racial  dif- 
ference in  the  fungus. 

To  determine  whether  various  cereals,  autoclaved,  influence  conidial 
length  differently,  plates  of  H.  No.  1  were  prepared  under  standard  condi- 
tions except  that  in  the  same  Petri  dishes  were  placed  shoots  of  wheat,  rye, 
barley  and  corn.  The  resulting  graphs  of  conidial  length  are  given  in  Fig. 
M.  The  differences  in  means  are  as  follows: 

On  rye  and  wheat,  0.40  ±  .29 
On  rye  and  corn,  0.45  ±  .20 
On  wheat  and  corn,  0.02  ±  .22 

The  mean  length  on  rye,  corn,  and  barley  is  in  close  agreement  with 
that  on  wheat,  and,  apparently,  under  these  conditions  the  species  of 
shoots  counts  for  little  in  its  influence  on  conidial  length. 

Conidial-length  graphs  (Fig.  N)  made  from  H.  No.  1  grown  on  fresh 
wheat-stems,  on  young  wheat  shoots,  on  wheat  leaves,  and  on  young  wheat 
plants,  all  autoclaved  in  test-tubes  with  a  few  centimeters  of  water,  show  a 
considerable  increase  over  those  under  standard  conditions  (Graph  42, 
Fig.  K) ;  also,  in  Graphs  58,  60,  and  61  (Fig.  N),  they  show  a  much  larger 
standard  deviation  and  coefficient  of  variability,  probably  due  to  the  va- 
riable humidity  under  these  conditions.  The  small  number  of  conidia 
measured,  and  the  lack  of  control  over  humidity  may  be  presumed  to  ac- 
count for  such  variation  as  is  seen. 

Live  wheat  inoculated  in  rag  doll  showed  at  the  6th  day  100%  infec- 
tion. These  infected  seedlings  were  placed  in  a  Petri  dish  on  moist  filter- 


123 

paper  and  the  conidia  allowed  to  develop  to  maturity.  Conidial  length 
here  (Graph  64,  Fig.  O)  was  somewhat  less  than  under  standard  conditions 
(see  Graph  42,  Fig.  K),  and  the  coefficient  of  variability  was  a  little  high. 

Conidial  breadth.- — H.  No.   1  was  quite  constant  in  conidial  breadth 
as  follows: 

M  (7  CV 

6.03  ±.04  0.55  ±.34  9. 13  ±.57 

The  ratio  of  conidial  length  to  conidial  breadth  is  an  important  one 
as  determinative  of  shape.     This  ratio  for  H.  No.  1  is  as  follows: 

mean  length  22.62  ±  .05   _ 

—    o  •  /  4   ^  .  Uo 


mean  breadth  6.03  ±  .04 

In  a  description  of  H.  No.  1,  written  in  May,  1919,  for  my  own  use, 
and  prepared  with  considerably  more  care  than  is  ordinarily  used  in  specific 
descriptions  of  fungi,  I  noted  the  conidia  as  3 — 8  septate  and  as  52.6 — 67.2 
XI 9. 2 — 24  IJL  long  on  wheat;  and  as  48 — 84X18 — 21.6  /JL  on  corn-meal 
agar,  whereas  my  more  extended  study  now  shows  the  mode  on  wheat  as 
78.2  /z,  the  mean  as  76.8  M»  and  the  range  from  34  to  98.6  /r,  the  breadth  as 
ranging  from  17  to  23.8  /-i,  with  the  mean  as  20.4  /*;  the  septa  with  a 
mode  of  8,  a  mean  of  7.9,  and  ranging  from  4  to  10.  I  may  here  note  also 
that  Bakke  (6)  in  his  description  of  H.  teres  gives  the  conidial  dimensions  as 
150  [or  105*]— 130X15— 20  ju,  and  the  septa  as  7— 14.  Thus  he  seems  to  have 
found  conidia  considerably  longer  than  I  did,  as  also  narrower  ones.  It 
should  be  said  that  the  data  obtained  by  this  study  of  graphs  of  H.  No.  1, 
though  involving  several  thousand  measurements,  fail  to  record  the  longest 
conidium  observed,  and  the  one  with  the  most  septa,  because  these  were 
both  seen  during  observations  which  rendered  their  inclusion  impossible; 
which  is  to  say  that  to  include  them  would  have  been  to  consciously 
select  these  unique  conidia  for  inclusion.  Anent  the  shortcoming  of  my 
own  brief  description  cited  above  may  be  quoted  the  Saccardian  de- 
scription of  H.  ravenelii:  "Spongiosum;  hyphis  flaccidis  flexuosis  nodosis 
ramosis,  inarticulatis;  conidiis  cymbiformibus,  3-4  septatis,  fuscis,  50 
ju  longis,  endo-chromotibus  isthmo  connexis."  Though  the  mode  is  approx- 
imately at  50-54  IJL  the  conidia  really  range  from  13  to  7lju  (see  Fig.  L). 
Very  similar  errors,  due  to  brevity  of  description,  exist  regarding  many 
or  all  known  species. 


*See  Pammel,  King,  and  Bakke  (90,  p.  180). 


124 
ETIOLOGY  OF  FOOT-ROT 

EVIDENCES    OF    ETIOLOGICAL    RELATION    OF    H.    NO.     1 

Constant  Presence  of  the  Pathogens 

In  all  cases  of  American  foot-rot  of  wheat  that  have  come  under  my 
observation  the  rotten  basal  portion  of  the  shoot  bore  and  was  to  a  large 
extent  occupied  by  a  mycelium,  which  grew  luxuriantly  within  the  wheat 
tissue  though  very  sparfngly  upon  its  surface,  coursing  lengthwise  within 
the  diseased  cells.  This  mycelium  was  hyaline,  septate,  vacuolate,  irregular 
in  thickness,  and,  in  short,  agreed  in  all  characters  with  those  of  H.  No.  1 
when  growing  in  rotting  wheat-tissue  (page  105). 

Absence  of  other  Constant  Parasites 

No  other  organism  which  might  be  considered  as  a  possible  parasite  was 
present  in  any  large  number  of  cases  in  or  on  the  wheat  tissue.  Amebae 
and  nematodes  were  present  in  great  numbers  in  the  soil,  but  appeared  to 
bear  no  relation  to  the  rot  of  the  wheat.  Various  fungi,  as  Fusarium  (two 
species),  Rhizoctonia,  Epicoccum,  Alternaria,  were  occasionally  found  on 
the  roots  or  stems,  but  each  only  rarely,  in  a  fraction  of  1%  of  the  cases, 
and  with  no  evidence  of  etiological  relation  to  the  rot  of  the  stem. 

Identity  of  Pathogene  proved  by  Culture 

Very  numerous  isolations  were  made  by  taking  bits  of  tissue  (1)  from 
diseased  sheaths,  (2)  from  diseased  stem-lesions,  and  (3)  by  stripping  away 
the  sheath,  disinfecting  the  remaining  surface  with  mercuric  chloride  and 
taking  out  diseased  bits,  with  precautions  against  contamination.  All 
such  diseased  bits  were  laid  on  the  surface  of  corn-meal  agar  plates.  Hun- 
dreds of  these  were  made,  with  the  result  that  in  practically  every  instance 
the  diseased  bit  gave  rise  to  Helminthosporium  conidia  in  general  aspect 
like  those  of  H.  No.  1.  Other  organisms,  as  mentioned,  occasionally 
occurred  on  these  plates,  but  in  only  a  small  per  cent,  of  instances.  It 
seems  entirely  conclusive  that  the  mycelium  constantly  found  in  the  rotting 
basal  portion  of  the  diseased  wrheat-stems  is  that  of  a  Helminthosporium. 

Evidence  of  Infectiousness 

Several  bags  of  soil  that  bore  diseased  wheat  in  1919,  near  Granite 
City,  Illinois,  were  brought  into  our  greenhouse  in  July,  1919.  In  this  soil 
was  planted  "Sultzer  Pride"  wheat,  and  the  planting  kept  liberally  watered. 
At  the  end  of  some  weeks  the  plants  were  removed,  and  on  examination  all 


125 

showed  browning  and  incipient  rot  of  the  basal  portion  of  the  stem.  Micro- 
scopic examination  and  agar  platings  from  these  stems  gave  results  identical 
with  those  stated  above.  One  plant  that  was  so  badly  rotted  in  the  pot 
as  to  fall  over  was  found  bearing  Helminthosporium  conidia  on  its  surface. 

Conidia  produced  in  Moist-chamber  Culture 

While  stems  with  diseased  lesions,  either  from  the  field  or  greenhouse, 
when  placed  in  an  ordinary  moist-chamber  rarely  gave  Helminthosporium 
conidia  (or,  if  they  did,  only  in  small  numbers),  if  the  diseased  stems  were 
placed  on  wet  filter-paper  in  moist  chamber  and  rather  closely  covered 
with  wet  filter-paper  Helminthosporium  conidia  invariably  developed  in 
quantity  on  the  lesions,  the  fungus  eventually  spreading  throughout  the 
available  wheat-tissue  and  producing  conidia  over  the  whole  surface 
(cf.  with  page  95). 

Evidence  from  Inoculation 

Severed,  live  wheat-shoots,  grown  under  aseptic  conditions,  were  placed 
as  under  standard  conditions  (Appendix,  page  180),  except  that  the  shoots 
were  not  autoclaved  but  put,  living,  upon  the  inoculated  agar.  All  such 
shoots  rotted  rapidly  and  completely,  the  shoot  being  eventually  covered 
by  Helminthosporium  conidia.  Since  direct  examination  showed  no 
contamination,  it  is  evident  that  H.  No.  1  can  cause  rot  of  the  wheat  tissue. 

To  determine  the  relative  rotting  power  of  this  organism  and  other 
Helminthosporiums  under  these  conditions,  fresh  aseptic  shoots  of  corn, 
wheat,  oats,  barley,  and  rye  were  laid  on  washed  agar  with  the  growing 
tip  toward  the  circumference  of  the  dish,  and  the  cut  end  in  contact  with 
the  outer  edge  of  the  spreading  mycelium  of  a  colony  about  5  cm.  in  diam- 
eter. These  were  examined  after  2  days  and  again  after  5  days,  and  the 
rate  of  browning  was  carefully  calculated.  In  this  way  seventeen  strains  of 
Helminthosporium  were  tested  as  to  their  ability  to  produce  rot  in  live, 
severed  cereal-shoots.  H.  No.  1,  the  foot-rot  organism,  showed  high 
rotting  ability,  completely  rotting  a  wheat  shoot  11  mm.  long  in  5  days, 
while  H.  No.  2  (H.  ravenelii)  produced  no  rot  on  any  cereal.  H.  No.  1 
rotted  corn  also,  but  much  less  rapidly  than  it  did  wheat,  and  its  rate 
on  oats,  barley,  and  rye  was  still  less.  Several  other  numbers  showed 
strong  rotting  power  on  wheat  shoots,  notably  H.  No.  10  (labeled  H. 
teres),  isolated  by  Dr.  Stakman  from  barley,  H.  No.  9  isolated  by  him 
from  wheat,  and  H.  No.  13  (labeled  H.  sativum],  isolated  by  Dr.  Durrell 
from  barley. 


126 

The  results  from  this  preliminary  work  indicate  also  a  very  wide  dif- 
ference in  the  susceptibility  of  these  cereals  to  rot  by  the  various  strains 
of  Helminthosporium.  Oats,  on  the  whole,  are  less  injured  by  them 
than  any  of  the  other  four  cereals  tested.  Corn  and  wheat  were  most 
often  first  in  susceptibility  to  certain  of  the  strains,  and  were  also  highly 
susceptible  to  more  strains  than  were  barley  and  rye. 

Seedlings  in  Petri  dishes  inoculated. — Aseptic  wheat-seedlings  were 
placed  on  moist  filter-paper  in  sterile  Petri-dishes  and  were  inoculated  in 
their  basal  region  in  three  ways:  by  placing  upon  them  ( 1)  wheat  tissue  rot- 
ted by  H.  No.  1  (pure  culture),  (2)  conidia  of  this  organism,  and  (3)  agar 
bearing  an  abundance  of  growing  mycelium.  No  difference  was  observed 
in  the  effectiveness  of  the  three  modes  of  inoculation.  Each  gave  a  100% 
infection,  always  visible  with  a  hand  lens  in  2  days  (Fig.  16)  as  a  small  spot, 
which  could  usually  be  seen  at  the  same  time  without  a  glass.  A  longer 
time  than  two  days  was  necessary  to  demonstrate  that  this  spot  would 
develop  into  a  general  rot,  but  so  it  did  in  all  cases  when  the  environ- 
ment was  favorable. 

Seedlings  in  rag  doll  inoculated. — Wheat  seedlings  with  shoots  2-3  cm. 
long  were  placed  in  a  special  form  of  rag  doll  (PI.  XXXIII)  and  inoculated 
with  H.  No.  1  by  placing  an  oese  of  conidia-suspension  on  the  base  of  each 
shoot  without  wounding.  Infection  was  apparent  to  the  naked  eye  in 
every  case  in  two  days,  and  the  results  in  six  days  are  shown  in  PI. 
XXXIV.  Rotting  occurred  in  6-12  days  under  favorable  conditions. 
At  6  days  the  roots  were  often  more  or  less  blackened  for  long  distances  and 
the  cortex  filled  with  mycelium.  Views  of  cross- sections  showed  a  heavy 
infection  of  the  second  leaf,  and  the  sheath  completely  occupied.  With 
excessive  moisture,  seedlings  were  killed  by  the  Helminthosporium  in  6 
days;  but  if  in  comparative  dryness,  only  small  lesions  resulted.  Seedlings 
similarly  placed  in  rag-doll  but  atomized  with  conidia-suspension  also  gave 
100%  infection,  and  the  infection  was  much  more  widely  distributed. 

Inoculation  by  diseased  tissue  or  by  fungus-bearing  agar  was  in  no  way 
superior  to  inoculation  with  conidia. 

Control,  or  check,  rag-dolls,  made  in  the  same  manner  but  without 
inoculum,  at  2  and  6  days  showed  no  lesions  even  under  microscopic  ex- 
amination. In  a  very  small  number  of  cases  there  was  infection  by  Hel- 
minthosporium in  the  checks,  and  in  a  few  instances  overgrowth  by  a 
Helminthosporium  similar  to  H.  No.  29,  with  geniculate  conidia. 

Roots  of  wheat  inoculated. — Conidia  of  H.  No.  1  were  placed  on  the  root- 
hairs  of  wheat-seedlings  in  rag  doll.  At  the  end  of  4  days  all  roots  so  in- 


127 


oculated  were  yellowish  or  pale  straw-color,  as  contrasted  with  the  white, 
uninoculated  roots,  and  they  had  scant  root-hairs.     Under  the  microscope 


FIG.  16. — Lesions  on  unwounded  wheat-seed- 
lings two  days  after  inoculation  with  conidia  of 
H.  No.  1.  The  shaded  portion  of  the  shoot  was 
yellow  to  brown. 


128 

the  cortical  tissue  was  seen  to  be  crowded  with  Helminthosporium  myceli- 
um coursing  mainly  in  the  longitudinal  direction  of  the  root.  The  mycelial 
threads  within  the  root  cortex  were  remarkably  thick — 13/z.  Wheat  seedlings 
2  cm.  long,  atomized  with  conidia  suspension  of  H.  No.  1,  in  6  days  were 
covered  with  infection  spots  over  their  whole  surface. 

Inoculations  in  soil. — Vials  12X70  mm.,  prepared  as  described  on 
page  180,  were  used  as  containers.  Wheat  seeds  were  germinated  asep- 
tically,  and  when  the  shoot  was  about  2  cm.  long  they  were  inoculated 
and  transferred  to  the  soil  in  a  vial.  The  results  differed  in  no  essential 
way  from  those  described  for  the  rag-doll  inoculations,  though  the  plant 
could  be  kept  longer  under  observation  since  it  was  not  solely  dependent 
on  the  seed  for  food.  Aseptic  wheat-grains  were  also  planted  in  these 
vials  with  the  inoculum  placed  in  three  different  positions:  (a)  on  the  seed; 
(b)  1.5  cm.  above  the  seed;  (c)  1.5  cm.  below  the  seed.  When  on  the  seed, 
lesions  occurred  low;  when  above  the  seed,  they  were  higher;  when  below 
the  seed,  no  lesions  were  on  the  stem  in  early  days  but  the  roots  were 
heavily  infected. 

Duplication,  in  pots  and  in  benches,  of  all  the  above  experiments 
made  in  vials  gave  identical  results. 

Recovery  of  Organism 

After  all  the  types  of  inoculation  mentioned  above,  the  organism  used 
in  the  inoculation  was  clearly  evident  in  the  tissues  and  producing  conidia 
upon  them,  and  by  dilution-plating  it  was  recovered  from  them.  During 
such  recovery  there  was  sometimes  evidence  of  bacterial  or  other  contami- 
nation, but  in  most  cases  of  each  type  of  inoculation  no  contamination 
occurred,  and  the  pathogenic  changes  noted  were  clearly  attributable  to 
the  fungus  used  in  the  inoculation. 

INFECTION    PHENOMENA    ON    WHEAT 

Conidia  of  H.  No.  1  and  of  H.  No.  14  when  placed  on  wheat  in  rag 
doll  germinated  from  one  or  both  ends  as  described  elsewhere.  The  germ- 
tube  grew  rapidly,  branching  freely,  and  oriented  itself  lengthwise  of  the 
shoot  more  frequently  than  crosswise  or  obliquely,  often  following  length- 
wise the  boundary  between  two  wheat-cells.  At  certain  places  where  this 
mycelium  touched  the  wheat-surface  it  swelled  slightly,  producing  a  round 
or  oblong  appressorium.  These  appresoria  sometimes,  probably  most  often, 
arose  by  the  simple  swelling  of  a  cell  of  the  main  thread  (Fig.  17),  though 
frequently  also  from  short  lateral  branches  (Fig.  17,  d)  or  where  the  terminal 


129 

cell  of  a  thread  abutted  against  the  wheat  tissue  (Fig.  17,  g).  So  far  as 
observed  they  differed  from  the  usual  mycelila  cells  only  in  shape.  The 
appressoria  are  very  numerous  (Fig.  17,  b).  They  are  usually  produced 
only  after  the  mycelium  has  grown  to  considerable  length;  not,  as  is  the 
case  with  some  fungi,  immediately  on  emergence  from  the  conidium.  In 


FIG.  17:  a,  H.  No.  1  on  wheat,  24  hours  after  inoculation,  showing  mycelium  arising  from  a  conidium, 
an  appressorium,  and  penetrating  mycelium;  b,  c,  d,  H.  No.  14,  showing  appressoria,  penetrating 
points,  and  "callus";  e,  f,  g,  h,  H.  No.  1:  e,  mycelium  within  cell,  and  with  a  penetrating  mycelium 
reaching  into  an  adjacent  cell,  a  "callus"  there  resulting;  /,  mycelium  ending  squarely  against  a  cell- 
surface,  penetrating  it  and  then  being  covered  by  "callus",  and  eventually  penetrating  this  and 
the  next  cell-wall,  the  latter  being  thickened;  g  and  h,  similar  to  e. 

most  cases  the  penetrating  mycelium,  viewed  from  above,  appears  as  a 
minute  bright  point,  or  as  if  a  minute  hole  had  been  pierced  in  the  wheat 
cell-wall,  much  as  is  seen  in  the  hyphopodia  of  Meliola  (82)  or  on  the 
appressoria  of  Gloeosporium  (64)  where  penetration  organs  arise.  Viewed 
laterally,  the  bright  point  of  the  appressorium  is  seen  to  mark  the  emer- 
gence of  a  haustorium-like  strand  which  I  shall  continue  to  call  the  pene- 
trating mycelium.  This  structure  is  much  thinner  than  the  usual  mycelium 
(see  Fig.  18)  and  of  different  staining  reactions.  It  penetrates  the  wheat 


130 


cell-wall,  and  is  sometimes  simple,  sometimes  branched.  At  the  place  where 
the  penetrating  mycelium  pierces  a  wall  and  enters  a  healthy  wheat-cell 
there  is  developed,  on  the  inside  of  the  wheat-cell  and  surrounding  and 
covering  the  penetrating  mycelium,  a  callus-like  structure  (Fig.  17,  e-g) 
which  for  brevity  I  shall  term  the  "callus".  As  the  penetratiftg^mycelium 
continues  to  grow,  the  "callus"  grows  pari  passu.  Where  many  penetrating 
mycelia  develop  near  each  other  this  "callus"  may  become  very  large 


L 


J 


FIG.  18. — H.  No.  1:  a,  large  "callus"-formation,  with  many  penetrating  mycelia  piercing  the 
cell  walls;  b,  mycelium  spreading  over  the  wheat  surface,  and  at  many  contact  points  producing 
appressoria  and  penetrating  mycelia;  c,  penetrating  mycelium  of  unusual  form,  and  the  "callus" 
rough. 

(Fig.  18,  a)  and  complicated.  The  "callus"  formation  seems  to  be  of  the 
nature  of  a  precipitation,  probably  resulting  from  toxic  action,  and  a  badly 
intoxicated  cell  can,  in  its  protoplasmic  disorganization,  make  numerous 
such  deposits  at  points  other  than  those  of  mycelial  entrance.  Thus  in 
some  instances  the  whole  inner  surface  of  a  cell's  walls  may  be  thickly 


131 

studded  with  small  dewy  drops,  apparently  of  precisely  the  same  character 
as  the  "callus."  (See  Fig.  23,  page  135.) 

The  host's  cell-wall  at  and  near  the  point  of  penetration,  is  markedly 
altered  chemically,  as  is  shown  by  various  stain-reactions.  Thus,  adjacent 
to  the  point  of  infection  several  different  regions  giving  different  chem- 
ical reactions  may  be  distinguished,  as  is  indicated  in  Fig.  19.  Region  3 
gives  the  usual  chlor-zinc-iodide  reaction  and  stains  like  normal  cellulose. 
None  of  the  other  regions  do  this.  Region  4  stains  darker  with  the  usual 


FIG.  19. — H.  No.  1 :  regions  of  a  young  diseased  spot: 
1,  mycelium;  2,  penetrating  mycelium;  3,  normal  wheat 
cell-Avail ;  4,  region  of  darker  staining ;  5,  region  of  lighter 
staining;  6,  diseased  inner  lamella;  7,  middle  lamella; 
8,  "callus." 

stains,  but  not  so  dark  as  normal  cell-wall.  The  "callus"  and  penetrating 
mycelium  stain  faintly  or  not  at  all.  The  middle  lamella  stands  out  clearly 
in  all  of  the  diseased  region,  and  on  each  side  of  it  the  inner  lamella  is  seen 
to  be  thickened  and  of  altered  stain-reaction.  Though  penetration  is  some- 
times directly  through  the  wall  it  is  much  oftener  into  the  middle  lamella, 
and  the  mycelium  shows  a  strong  tendency  to  follow  along  the  line  of 
division  between  two  cells,  thus  giving  a  gridiron  effect  to  the  mesh.  This 
is  possibly  due  to  chemotropic  attraction  by  the  middle  lamella  or,  possibly, 
because  this  is  the  weakest  place  in  the  cuticle.  No  case  of  stomatal 
entrance  was  observed;  indeed,  on  the  sheaths  of  "Golden  Chaff"  wheat 
stomata  are  seldom  present. 

Once  within  the  host  cell  the  mycelium  grows  rapidly,  soon  nearly  or 
completely  filling  it  (Fig.  20),  and  often  forming  a  mass  so  dense  that  it 
resembles  a  pseudoparenchyma.  Both  longitudinal  and  transverse  sections 


132 

show  clearly  that  the  mycelium  is  within,  not  between,  the  host  cells. 
Penetration  into  adjoining  live  cells  is  attended  by  the  same  phenomena 
of  penetrating  mycelium,  ' 'callus"  formation,  and  wall-changes,  though 
appressoria  were  not  observed  in  such  cases,  possibly  on  account  of  the 
difficulty  of  observation.  Penetration  into  dead  cells  is  not  attended  by 
these  phenomena. 

The  chronological  history  of  a  lesion  from  a  simple  infection  begins 
with  the  attack  on  one  cell,  which  is  soon  overcome  and  occupied,  and  at 
24,  or,  better,  48  hours  after  inoculation,  observation  with  a  16  mm. 
objective  shows  regions  with  one  to  several  cells  diseased  and  browned, 


FIG.  20. — H.  No.  1  on  wheat:  a,  mycelium  in  cells  and  penetrating  the  side  walls;  b,  mycelium 
running  lengthwise  within  the  wheat  cells. 

and  the  protoplasts  undergoing  disorganization  and  becoming  browned. 
Owing  to  the  length  of  the  wheat-cells,  the  diseased  regions  are  much 
longer  than  broad,  and  in  many  instances  two  diseased  cells  or  two  rows 
of  them  are  seen  with  a  quite  healthy  cell  between  them  (Fig.  21).  Under 
action  of  Javelle  water  the  healthy  cells  plasmolize  beautifully,  while  the 
sick  cells  show  no  plasmolysis.  Treated  with  acid  fuchsin  in  glycerine, 
normal  cells  show  no  stain,  while  in  diseased  cells  the  entire  protoplast 
becomes  pink  and  the  inner  lamella,  which  is  swollen,  also  stains  pink. 
This  softening  and  swelling  of  the  lamellae  was  extensively  studied  by 
de  Bary  (8),  Ward  (123)  and  Biisgen  (30).  De  Bary,  who,  in  1886,  was 
first  to  separate  .a  cytolytic  enzyme  from  fungi  (Sclerotinia  libertiana),  states 
that  as  the  inner  lamellae  undergo  partial  dissolution  they  continue  for 
a  time  to  give  the  cellulose  reaction,  but  eventually  swell,  disorganize,  and 
lose  this  property  (8,  page  420) .  He  also  describes  the  fungus  as  growing  in 
the  middle  lamella.  Ward  (123)  describes  the  cellulose  as  swelling  and  soft- 
ening under  action  of  the  enzyme  produced  by  Botrytis.  Here,  too,  the 


133 


mycelium  grows  in  the  middle  lamella.  Jones  (71),  working  with  Bacil- 
lus carotovorus ,  reports  that  the  enzyme  produced,  attacks  more  strongly 
the  middle  lamella,  but  he  noted  also  a  softening  and  swelling  of  the  inner 
lamella,  but  found  that  the  cellulose  stains  (e.  g.,  chlor-zinc-iodide)  "give 
clear  blue  reactions  with  these  fully  softened  walls."  Van  Hall  (63), 
working  with  Bacillus  omnivorus  on  Iris,  reports  a  similar  condition.  The 
inner  lamellae,  swollen  by  Helminthosporium,  no  longer  react  as  cellulose 
under  this  test.  Blackman  and  Welsford  (18),  who  describe  in  detail 
the  entrance  of  Botrytis  cinerea  into  bean  leaves,  state  that  neither  before 
nor  after  penetration  did  the  staining  reactions  of  the  cuticle  give  any 
evidence  of  its  being  softened  or  swollen  or  in  any  way  altered  chemically 
(though  the  subcuticular  walls  usually,  if  not  always,  swell),  and  no  swelling 


FIG.  21.  —  H.   No.   1  on  wheat  shoots,  second  day  after  inoculation. 
Shaded  portion  was  colored  brown. 

of  the  subcuticular  cellulose  was  observed  before  the  passage  of  the  invad- 
ing hypha  through  the  cuticle.  Pathogenic  changes  in  the  inner  lamella 
precede  those  in  the  protoplast,  that  is,  no  toxin  acts  upon  the  proto- 
plast prior  to  the  swelling  of  the  lamellae.  The  subcuticular  layer  swells. 
Penetration  of  the  cuticle  is  by  pressure.  Gardner  (58)  mentions  no 
changes  occurring  normally  in  staining  reaction  of  host  cellulose  in  leaves 
attacked  by  Colletotrichum,  though  in  cases  of  delayed  penetration 
he  notes  that  the  cell-wall  under  the  appressorium  retained  safranin  bet- 
ter than  did  normal  cell-walls.  In  fruit  penetration,  however,  he  found 
that,  characteristically,  the  inner  lamella  was  so  altered  as  to  retain  saf- 
ranin. Ihe  action  appears  to  be  different  in  both  quality  and  quantity 
from  that  described  by  Newcomb  (86),  who,  studying  enzymes  in  seeds, 
states  that  "with  all  the  ferments  the  wralls  at  first  become  hyaline,  appear 


134 

gradually  more  transparent  and  finally  'melt  away.'  '  In  Colletotrichum 
Gardner  (58)  found  the  fungus  similarly  seeking  the  "depressions  bounding 
the  epidermal  cells."  This  place  of  entrance  is  characteristic  of  many  fun- 
gi—see Biisgen  (30),  Rehrens  (15),  Ward  (123),  Noack  (ST^Miyoshi  (83), 
Nordhausen  (88),  Schellenberg  (99),  and  Aderhold  (1).  The  last  three 
named,  believe  this  to  be  due  to  chemotaxic  influences.  Noack  in  describ- 
ing the  entrance  of  H.  gramineum  into  the  host  mentions  the  appressoria. 
Similar  structures  have  also  been  described  in  the  anthracnose  fungi  by 
Hasselbring  (64)  and  by  Gardner  (58),  the  pore  in  these  structures  being 
such  as  I  find  in  Helminthosporium,  though  the  appressorium  in  the  anthrac- 
nose fungi  is  a  mere  swelling  and  is  hyaline.  Similar  extreme  narrowness 
of  the  mycelium  at  the  actual  point  of  penetration  of  host-walls  is  shown 


FIG.  22. — H.  Xo.   14  on  wheat,  showing  fan-like  mode  of  branching,  see  p.  105. 


also  by  Ward  (123,  fig.  57),  Gardner  (58,  page  27),  Hasselbring  (64*, 
Biisgen  (30),  and  Noack  (87).  Bakke  (6)  says  of  H.  teres  that  the 
mycelium  "penetrated  the  epidermis  directly  and  made  its  way  through 
the  intercellular  spaces,"  but  he  gives  no  further  details. 

Conditions  very  closely  resembling  the  "callus"  formation  are  figured 
by  Dastur  (41.  figs.  8,  9);  depicting  the  entrance  of  smut  into  sugar- 
cane. This  appears  to  have  occurred  only  occasionally,  and  Dastur  re- 
gards the  "callus"  ("plug")  as  probably  a  means  of  preventing  infection. 
Conditions  somewhat  resembling  that  of  the  "callus"  formation  are  de- 
scribed and  figured  by  Wolff  (128,  figs.  2,  3)  and  by  Brefeld  (25,  fig.  2) 
in  the  penetration  of  smuts  into  cereal  tissue.  Wolff  (128,  p.  20)  de- 
scribing this  says:  "Es  tritt  hierbei  der  eigenthiimliche  Umstand  ein, 


135 

dass  der  Faden,  sobald  seine  Spitze  in  das  Innere  der  Zelle  tritt,  nicht 
frei  in  dieses  hineinwachst,  sondern  von  den  inneren  Schichten  der  Zell- 
wand,  welche  sich  gleichsam  aus  stiilpen,  wie  in  eine  Scheide  von  bald 
grosserer  bald  geringerer,  oft  sehr  betrachtlicher  Starke  eingeschlossen 
wird  und  in  dieser  bis  zur  nachsten  Zellwand  weiter  wachst."  Brefeld 
describes  very  similar  conditions,  including  much  thickening  which  is 
of  yellow  color,  but  instead  of  interpreting  it  as  an  enclosing  sheath  he 
regards  it  as  wholly  due  to  thickening  of  the  walls  of  the  mycelium  itself. 
He  moreover  states  that  this  phenomenon  is  indicative  of  conditions 
in  the  host,  as  too  great  age,  that  are  unsuitable  to  infection,  and  that  it 
is  not  in  evidence  when  the  host  is  in  fully  susceptible  condition.  Which- 
ever may  be  the  true  interpretation  in  the  case  of  cereal  smuts,  I  am 
convinced  that  in  case  of  Helminthosporium^the  "callus"  is  produced  by 


FIG.  23. — Infection  by  H.  No.  1,  24  hours  after  inoculation,  showing 
thickening  of  the  wheat  cell-walls  by  deposition  on  their  inner  surfaces. 
(Text  citation  at  top  of  p.  131.) 

the  wheat-cell,  and  is  not  part  of  the  mycelium.  Ravn  (91),  describing 
the  reactions  to  the  intercellular  mycelium  of  Helminthosporium  in  cereals, 
states  that  a  thickening  appears  upon  the  cell-wall  of  the  host,  resembling 
a  drop  segregated  from  the  cell,  and  that  several  such  thickenings  may  be 
seen  upon  one  cell,  sometimes  filling  the  intercellular  spaces  completely. 
They  seem  to  differ  from  those  that  I  describe  (Fig.  17),  however,  in  posi- 
tion, since  they  are  without,  not  within,  the  cell,  and  in  composition,  as 
those  noted  by  Ravn  take  aniline  stains  readily. 

Ravn  (91,  fig.  23)  describes  an  appressorium  very  much  like  that 
which  I  find  and  states  that  the  mycelium  from  it  enters  the  epidermal  cell, 
where  it  so  increases  that  it  may  fill  the  cell ;  then  makes  its  way  to  the 
intercellular  spaces  and  grows  there  exclusively,  never  again  entering 
any  of  the  cells  even  by  means  of  haustoria.  It  therefore  appears  from 
his  statements  and  figures  that  the  Helminthosporiums  with  which  he 


136 

worked,  differed  in  a  very  fundamental  way,  as  pathogenes,  from  those 
which  I  am  studying,  his  forms  being  intracellular  (except  as  regards 
the  first  cell  invaded),  and  not  at  once  killing  the  adjacent  cells.  That 
is,  the  condition  pictured  is  much  like  that  presented  by  Albugo,  Perono- 
spora,  Puccinia,  etc.,  except  for  the  absence  of  haustoria.  The  forms 
with  which  I  deal,  on  the  other  hand,  though  they  enter  through  the  middle 
lamellae,  immediately  become  intracellular  and  at  once  kill  the  protoplast 
of  the  invaded  cell,  and  proceed  similarly  with  other  cells.  These  differing- 
conditions,  if  substantiated  by  further  study,  probably  indicate  funda- 
mental differences  in  the  fungi  in  regard  to  their  production  of  toxins  or 
enzymes,  and  certainly  indicate  an  entirely  different  type  of  pathogenicity. 
In  these  early  stages  the  disease  is  properly  a  spot  and  not  a  rot.  Whether 
it  will  develop  into  a  true,  general  rot  depends  upon  conditions.  Phenomena 
like  those  described  under  the  present  heading,  though  differing  in  de- 
tail, were  noted  with  H.  Nos.  6,  8,  9,  14,  21,  36,  39,  40,  and  41. 

Action  of  various  strains  of  Helmintkosporium  on  wheat  shoots. — Tests 
in  rag  doll,  at  medium  moisture,  with  H.  No.  1  and  H.  No.  3  gave  at  2 
days  100%  infection  for  both;  at  6  days  there  was  no  appreciable  difference 
between  the  two;  while  at  10  days  all  shoots  were  rotten  under  H.  No.  1 
and  some,  but  not  so  many,  under  H.  No.  3.  The  test  was  repeated  with 
14  strains  of  Helminthosporium.  All  strains  at  2  days  showed  100% 
infection;  the  controls,  no  infection.  The  infection  phenomena  with  all  of 
these  strains  were  all  of  the  character  described  on  pages  128,  129,  showing 
penetrating  mycelium,  "callus,"  etc.  At  6  days  H.  Nos.  1,  4,  5,  8,  13-16,  20, 
and  21  had  all  produced  some  rot.  The  roots  also  were  distinctly  yel- 
lowed by  H.  Nos.  15  and  16,  while  H.  No.  20  showed  less  rotting  than 
the  other  numbers  mentioned  above.  H.  Nos.  29  and  39  produced  no 
rotting,  and  the  lesions  were  visible  only  through  a  lens,  but  thus  viewed, 
showed  100%  infection,  as  indicated  by  the  usual  infection  phenomena. 
H.  Nos.  3,  6,  9,  17,  and  18  remained  local,  as  at  2  days.  H.  No.  29,  a 
Helminthosporium  with  geniculate  conidia,  germinated  abundantly  from 
both  ends  of  the  conidium,  and  on  wheat  produced  many  penetrating 
mycelia  and  an  abundant  mycelium  within  the  host,  though  the  mycelial 
invasion  reached  only  a  few  cells,  and  while  extending  for  a  considerable 
distance  lengthwise,  made  but  little  progress  laterally.  The  appressoria 
were  usually  pyriform,  as  was  also  the  penetrating  mycelium,  differing 
thus  from  H.  No.  1  (Fig.  17).  Similar  tests  were  made  with  three  saltants, 
M6,  M8,  and  M38.  Notes  at  2  days  showed  100%  infection,  and  at  6 
days  much  rot  by  M6,  and  considerable  rot  by  the  other  two. 


137 

Though  infection  can  be  determined  with  certainty,  I  have  as  yet  no 
means  of  accurately  measuring  rotting  power,  or  of  determining  whether 
differences  noted  in  rotting  are  due  to  environment,  host,  or  fungus. 
It  seams  clear,  however,  that  H.  No.  29  is  capable  of  causing  only  local 
spotting;  and  that  the  other  numbers,  perhaps  even  the  saltants,  vary 
somewhat  among  themselves  in  rotting  capacity,  most  of  them  causing  rot- 
ting to  some  extent  under  favoring  conditions. 

The  fact  that  so  many  and  diverse  races  of  Helminthosporium  are 
able  to  cause  rot  of  wheat,  led  me  to  test  the  ability  of  Alternaria  to  para- 
sitize wheat  seedlings.  An  Alternaria,  found  commonly  on  wheat  seed,  was 
isolated  and  inoculation  made  in  rag  doll  on  wheat  seedlings.  At  24  hours 
many  wheat  cells  showed  diseased  spots,  being  in  every  way  like  those 
described  on  pages  128,  129,  including  the  swollen  middle  and  inner  lamellae, 
browning  of  the  cell-contents,  and  formation  of  the  "callus"  and  penetrating 
mycelium.  The  Alternaria  mycelium  crossing  several  middle  lamellae, 
usually  produced  an  appressorium  and  penetration  at  each  middle  lamella. 
The  Alternaria  mycelium  was  also  seen  to  enter  the  wheat-cells,  killing  a 
few  of  them,  but  in  no  instance  was  this  fungus  observed  to  cause  rotting 
or  to  produce  a  spot  large  enough  to  be  visible  to  the  naked  eye.  It  was 
seen,  however,  to  penetrate  the  cells  of  the  root  cortex  quite  extensively, 
causing  a  slight  browning.  Sterigmatocystis,  Penicillium,  and  several 
other  fungi  supposed  to  be  mere  saprophytes,  were  treated  in  simitar 
manner,  but  produced  none  of  the  phenomena  of  infection. 

SUSCEPTIBILITY    OF    VARIOUS    HOSTS    TO    INFECTION 

Tests  in  rag  doll  with  H.  No.  1 

Corn. — Three  seedlings  showed  no  infection  at  2  days,  though  conidia 
were  present  and  had  germinated.  At  6  days  all  three  plants  were  in- 
fected, the  infection  being  confined  to  one  or  two  cells,  though  the  myce- 
lium was  clearly  evident  in  these.  Pammel,  King,  and  Bakke  (90)  report 
negative  results  regarding  infection  trials  of  H.  sativum  on  corn,  but  their 
tests  were  limited  to  leaves. 

Barley. — At  two  days  one  plant  was  slightly  infected,  showing  sev- 
eral lesions.  In  these  the  mycelium  was  abundant  within  the  cells.  Eight 
were  not  infected.  At  6  days  the  infection  showed  no  further  progress. 

Rye. — At  two  days  three  plants  were  infected;  six  not  infected.  At 
6  days  the  infection  showed  no  progress.  The  mycelium  was  observed  with- 
in the  cells  and  infection  phenomena  were  as  on  wheat. 


138 

Sorghum  (Holcus  sp.). — There  was  100%  infection  of  both  roots 
and  stems,  with  pronounced  rot.  T  he  same  phenomena  were  observed  as  on 
wheat,  including  the  appressoria,  penetrating  mycelium,  and  "callus." 
The  sap  of  the  infected  cells  was  strongly  tinged  with  red,_and  the  ' 'cal- 
lus" and  appressorium  were  deep  red.  Adjacent  colorless  walls  soon  be- 
came swollen  and  reddened.  The  red  coloring-matter  is  absorbed  by  the 
nucleus,  which  becomes  as  brilliantly  colored  as  by  an  aniline  dye.  At  six 
days  the  shoots  and  roots  were  heavily  infected,  the  diseased  regions 
assuming  a  deep  red,  almost  black,  color,  and  conidia  formed  abundantly 
over  the  lesions.  When  such  specimens  were  placed  in  alcohol,  the  red 
color  diffused  to  the  alcohol,  coloring  it  strongly.  This  red  coloration 
by  the  host  is  a  response  common  on  invasion  of  either  bacteria  or  fungi 
on  the  sorghums  and  sugar-cane,  and  on  corn  in  the  case  of  some  diseases. 

Sudan  grass  (Holcus  sorghum  sudanensis) . — At  ten  days  1  seedling 
gave  positive  and  5  gave  negative  results;  at  six  days,  5  positive  and  4 
negative.  Infection  was  slight  on  a  few  cells,  but  the  mycelium  was  evident 
within  the  cells,  and  infection  phenomena  as  on  wheat  were  observed. 

Common  millet  (Chaetochloa  italica). — At  two  days  10  gave  positive 
results.  At  six  days  the  rot  was  progressing  into  the  roots  faster  than 
into  the  stem,  though  black  spots  3 — 4  cm.  long  were  apparent.  Infection 
phenomena  were  observed  as  on  wheat,  and  much  mycelium  was  seen 
within  the  tissue. 

German  millet  (Chaetochloa  italica  germanica). — The  results  were  prac- 
tically the  same  as  with  common  millet. 

Amber  cane  (Holcus  sorghum]. — Results  were  much  as  on  sorghum. 

Red  top(Agrostis  palustris). — No  phenomena  of  infection  were  observed . 

Beans. — No  rot  was  produced,  no  "callus",  nor  any  other  of  the  usual 
signs  of  infection;  nor  was  it  certainly  determined  that  the  mycelium 
entered  the  host-cells,  though  it  seems  probable  that  the  fungus  killed 
some  of  the  bean  cells. 

Inoculation  of  leaves. — Pots  of  well-established  seedlings  of  wheat, 
oats,  rye,  barley,  corn,  German  and  common  millet,  and  sorghum  were 
placed  in  a  humid  atmosphere  (above  90%  relative  humidity)  and  atomized 
with  suspension  of  H.  No.  1  conidia.  Well-defined  spots  occurred  fre- 
quently on  barley,  less  frequently  on  wheat.  Leaf-spots  due  to  a  Helmin- 
thosporium,  apparently  H.  No.  1,  also  occurred  naturally  on  wheat  in  the 
greenhouse.  Such  spots  were  first  pale;  later  with  a  mummified  dark 
center  surrounded  by  a  pale  zone;  and  were  oval  in  outline.  In  rye  the 
mycelium  was  seen  to  be  abundant  within  the  cells,  and  complete  death 


139 

of  the  affected  leaf,  and  also  rotting  without  spotting,  resulted.  On  the 
leaves  in  the  humid  air  of  the  rag  doll  occasional  spontaneous  infections 
were  noticed.  In  such  cases  the  infection  rapidly  spread,  involving  nearly 
all  of  the  leaf,  which  first  turned  pale,  then  very  slightly  brown.  Aerial 
mycelium  and  conidia  were  profuse  over  the  diseased  portion. 

SUMMARY    CONCERNING    ETIOLOGY    OF    FOOT-ROT 

The  evidence  is  conclusive  that  Helminthosporium  is  the  cause  of 
the  basal  rot  of  the  wheat-stems.  It  is  the  only  parasite  constantly 
present,  and  has  been  repeatedly,  and  by  many  methods,  proved  capable  of 
causing  such  rot.  This  conclusion  is  in  accord  with  the  findings  of  Beck- 
with  (14),  who  as  early  as  1911  showed  that  Helminthosporium  is  a  very 
common  parasite  within  the  tissue  of  wheat-plants.  Bakke  (6)  in  1912 
reported  that  when  conidia  of  H.  teres  were  placed  on  barley  seeds, 
"At  the  end  of  two  weeks'  time  there  were  not  over  seven  seedlings  to 
the  row  [originally  there  were  twenty-five].  The  roots  were  not  in  any 
sense  indicative  of  a  healthy  state  of  growth."  Oats  and  fescue-grass 
were  not  susceptible.  A  seedling  blight  of  wheat  observed  since  1910  has 
been  described  by  Stakman  (113)  in  Minnesota,  where  in  1918-19  it  became 
seriously  injurious.  The  symptoms  include  dwarfing,  foot-rot,  and  root- 
rot.  The  disease  appears  to  be  closely  like,  if  not  quite  identical  writh, 
the  one  which  is  the  subject  of  this  paper.  She  proves  conclusively  that 
the  cause  is  a  Helminthosporium.  A  foot-rot  of  wheat  due  to  a  Hel- 
minthosporium having  quite  different  morphological  characters  is  also 
known  in  Sudan  (see  No.  46,  page  184). 

Certain  of  Ravn's  experiments  (91)  conducted  by  inoculating  seeds 
on  wet  filter-paper  in  a  Petri  dish,  gave  conditions  much  like  those  in 
the  rag  dolls.  He  makes  no  mention,  however,  of  infection  of  the  sheath 
nor  of  the  occurrence  of  "rotting  of  the  basal  region. 

II.     Evidence  and  Discussion  of  the  Occurrence  of 
Saltation  within  the  Genus  Helminthosporium 

INTRODUCTORY 

Early  in  my  study  of  this  Helminthosporium  of  foot-rot  of  wheat 
(herein  designated  as  H.  No.  1)  it  was  noted  that  occasionally  certain  sectors 
of  a  colony  growing  on  an  agar  plate  differed  more  or  less  from  the  rest  of 
the  colony  (PI.  XXII,  5;  PI.  XXIII,!).  This  phenomenon  is  of  rather 
common  occurrence  in  work  involving  Petri-dish  cultures  of  either  fungi  or 
bacteria,  and  little  significance  was  at  first  attached  to  it;  but  later,  when 


140 

the  frequent  recurrence  of  these  variant  sectors  commanded  attention,  trans- 
fers were  made  from  several  of  them  to  freshly  poured  agar-plates,  and  a 
transfer  from  the  normal  portion  of  the  colony  was  added  to  each  of  these 
plates  at  a  distance  of  about  2  cm.  from  the  other  transfer.  The  variant 
transfer  was  then  marked  on  the  bottom  of  the  doubly  occupied  plate  as 
M  (indicating  mutant),  and  the  normal  transfer  as  O  (indicating  original). 
In  all  the  early  transfers  the  M  transfer  resulted  in  a  colony  (Ml)  of  decid- 
edly slower  growth  and  more  profuse  conidial  production  than  that  pro- 
duced by  the  O  transfer.  The  two  colonies  also  differed  markedly  in  general 
appearance  owing  to  minute  single  differences  which  were  often  difficult  to 
analyze,  but  which  in  the  aggregate  constituted  distinctions  which  were  so 
well-marked  and  obvious  that  at  first  sight  one  would  say  that  the  two 
colonies  were  those  of  two  distinct  fungi  (PI.  XXIII,  lower  fig. ;  PI.  XXVII). 
When  these  colonies  grew  to  fill,  or  nearly  to  fill,  the  plate,  transfers  from 
them  were  made  to  new  agar  plates,  and  later,  transfers  from  these  second 
plates,  and  so  on,  the  series  of  transfers  being  a  long  one.  It  was  found 
that  the  differences  appearing  in  the  Ml  and  Ol  colonies  were  usually 
maintained  on  succeeding  plates.  These  findings  led  to  the  tentative 
assumption  that  forms  in  the  variant  sectors  were  mutants  or  saltants  of 
a  more  or  less  permanent  nature,  and  a  more  serious  study  of  this  phe- 
nomenon was  undertaken. 

In  their  origin  the  variants  or  Saltants  always  appear  as  sectors  which 
differ  from  the  portion  of  the  parent  colony  adjacent  to  them  (see  PI.  XXII 
— XXV) .  To  the  naked  eye  the  most  common  deviations  from  the  orig- 
inal type  are  in  density,  color,  and  rate  of  growth.  Closer  observation,  with 
the  microscope,  frequently  shows  variation  in  the  grouping,  size,  and  shape 
of  the  conidia,  and  in  the  branching  of  the  mycelium.  Quite  often  many 
small  sectors  of  divergent  character  appear  at  the  edge  of  a  large  colony, 
especially  on  a  plate  that  is  beginning  to  dry.  Many  of  these  divergencies 
are  merely  modifications  due  to  local  environmental  changes,  and  whether 
they  are  more  can  be  determined  only  by  close  study  of  their  behavior  in 
subsequent  transfer  or  transfers.  Closer  consideration  of  the  characters 
involved  in  these  saltations  is  best  deferred  to  the  following  topic. 

In  following  this  discussion  it  must  be  borne  in  mind  that  M  refers  to 
the  variant  sector  on  the  plate  on  which  it  originated;  Ml,  to  the  colony  re- 
sulting from  the  first  transfer  from  M;  M-2,  to  that  resulting  from  the  first 
transfer  from  Ml,  and  so  on;  and  that  O  refers  to  the  original  colony  in 
which  the  M  arose.  It  is  my  custom  to  give  the  saltant  a  serial  number 
(writing  this  on  the  plate  in  which  it  was  found),  and,  usually,  to  transfer 


141 

both  the  saltant  and  the  original  to  the  same  plate  so  that  they  may  have 
the  same  environmental  conditions,  the  identical  quantity  and  quality  of 
agar,  and  by  growing  close  together  may  render  comparison  easy.  Notes 
on  the  origin  and  subsequent  behavior  of  the  saltants  were  made  under  the 
eerial  number,  ard  the  transfers  were  designated  by  additional  numbers 
Thus,  IVI98-7  refers  to  saltant  No.  98,  transfer  7. 

CHARACTERS  OF  SALTANTS  AS  SHOWN  IN  TRANSFERS 

General  appearance. — The  colonies  of  the  saltant  and  of  the  original 
when  grown  on  the  same  plate  were  usually  so  strikingly  different  in  gen- 
eral appearance  (Fl.  XXII,  XXIII)  that  a  mere  glance  sufficed  to  give 
the  impression  that  they  were  colonies  of  two  different  species.  This  dif- 
ference in  general  appearance  is,  on  analysis,  referable  to  one  or  more  of 
the  individual  differences  mentioned  below. 

Pate  cf  linear  growth. — Frequently  the  saltant  was  of  much  slower 
growth  than  the  original,  resulting  in  an  M  colony  of  much  less  diameter 
than  that  of  the  O  colony,  being  often  less  than  half  of  it  (see  two  ex- 
amples: one  given  in  Fl.  XXII  and  one  in  PI.  XXIII).  In  some  instances, 
however,  the  M  colony  grew  faster  than  the  O  colony. 

Ccnidial  production. — Frequently  the  M  colony,  especially  when  slow- 
growing,  was  much  more  productive  of  conidia  than  the  O  colony,  so  much 
so  as  to  give  the  colony  a  decidedly  perceptible  darker  color.  In  several 
instances,  however,  the  M  colony  was  of  the  opposite  character,  producing 
few  conidia  or,  in  some  cases,  going  to  the  extreme  of  appearing  to  pro- 
duce none  at  all.  Generally  speaking,  rate  of  linear  growth  was  in  inverse 
ratio  to  that  of  conidia-production ;  while  those  saltants  that  were  pale  and 
possessed  much  aerial  mycelium  were  usually  of  rapid  linear  growth  and 
low  conidial  production. 

Ccnidial  clusters. — Some  saltants  varied  strikingly  from  each  other 
and  from  the  originals  in  the  mean  number  of  conidia  borne  per  conidio- 
phore. 

Ccnidial  length,  breadth,  septation,  and  shape. — These  characters,  as 
evidenced  by  casual  observation  or  by  a  study  of  graphs  and  the  data 
derived  from  them,  are  shown  to  be  strikingly  different  in  various  saltants. 
For  clearness  I  present  in  this  connection  records  concerning  only  a  few 
saltants,  giving  graphs  and  data  for  others  later. 

Graphs  of  ccnidial  length  of  saltants  M35,  M36,  and  M40,  those  which 
show  greatest  deviation  from  originals  in  this  regard,  are  given  in  Fig.  P 
with  the  essential  data.  It  is  to  be  observed  that  the  modes  of  M35  and 


142 

M40  are  57.8  and  64.6  n,  respectively,  far  below  the  mode  of  the  original, 
which  was  81.6  M-  M36  shows  less  striking  difference,  but  this  is  still 
marked.  Comparison  of  the  means  shows  those  of  M36  and  M40  to  be 
approximately  17  and  18 -divisions  (1  division  =  3. 4  /*),  while  the  original 
was  23  divisions.  In  other  words,  the  conidia  of  M36  wese-only  about 
three  fourths  the  length  of  the  normal  conidium  of  H.  No.  1.  Such  dif- 
ferences as  they  appeared  in  the  microscope  are  shown  in  PL  XXVI.  The 
difference  in  variability  is  also  strikingly  large. 

Striking  variation  in  conidial  breadth,  both  relative  and  absolute,  was 
observed.  Graphs  and  data  of  the  more  pronounced  cases  are  presented 
in  Fig.  Q  and  others  are  given  later.  In  connection  with  Fig.  Y  (Graphs 
114-138)  are  given  summary  data  on  the  conidial  length  of  saltants  in- 
cluded in  this  study.  It  is  to  be  noted  (Graph  6A,  Fig.  B)  that  whereas 
the  mode  of  the  ordinary  conidium  stood  at  20.4  /i  and  no  conidia  exceeded 
a  thickness  of  23.8  /z,  the  modal  thickness  of  M8-7  (Graph  75,  Fig.  Q)  is 
23.8  ju,  with  many  conidia  27.2  ^  in  thickness,  one  even  30.6  /z.  Such  dif- 
ferences between  saltants  and  the  parental  form  are  presented  to  the  eye 
in  PI.  XXVI. 

The  ratio  of  conidial  length  to  conidial  breadth  is  perhaps  still  more 
striking  than  the  mere  variation  in  length.  In  such  variants  as  M6  (PI. 
XXVI,  b)  and  M8,  while  increased  greatly  in  thickness  the  conidia  were 
at  the  same  time  absolutely  shorter,  thus  emphasizing  to  the  eye  both 
differences.  The  ratio  of  length  to  breadth  in  H.  No.  1  is  as  follows: 

mean  length       _   22.62  =*=  .05       _  # 

mean  breadth  6.03  ±  .04 

while  in  a  sample  of  one  of  its  saltants  this  ratio  is 

mean  length       _   20.67  ±  .22  ^ 

mean  breadth  7.82  =*=  .11 


and  in  another  sample  of  the  same  saltant  it  is 
mean  length  19.58  ±  .30 


mean  breadth  7.30  =t  .06 


2.67   ±   .05  = 


*Probable  error  was  computed  according  to  the  above  formula  kindly  furnished  me  by  Dr.  J.  A.  Det- 

r> 

lefsen,  where  a  <=  probable  error  of  A;  b  =  probable  error  of  B;  and  E  =  probable  error  of  —  • 

A 


143 

Variations  in  septation  were  also  noted;  thus  in  Fig.  R,  Graphs  79-82 
are  quite  different  from  Graphs  83  and  84,  while  all  of  these  are  lower  than 
the  results  gotten  from  H.  No.  1  in  Graph  35,  Fig.  J.  I  attach  but  little 
value,  however,  to  these  variations  because  they  seem  inconstant. 

Variation  in  conidial  shape  is  common,  some  saltants  showing  the 
sides  more  nearly  parallel  than  others,  and  the  conidium  as  a  whole  less 
elliptical  or  fusiform. 

Variation  in  submerged  mycelium. — Aside  from  rate  of  growth  and 
variation  in  branching  which  resulted  in  changes  in  density  of  the  colony, 
differences  in  the  submerged  mycelium  were  observable  in  but  two  cases, 
most  strikingly  so  in  M26.  In  this  saltant  certain  hyphal  threads  near 
the  edge  of  the  colony  appeared  to  be  much  more  vigorous  than  their 
neighbors,  becoming  a  trifle  thicker,  and  lengthening  with  such  rapidity 
as  far  to  outstrip  the  others,  reaching  out  as  single  strands  to  a  consider- 
able distance  beyond  the  usually  even  frontier  of  the  colony,  beginning 
then  a  dense,  bushy  branching  in  all  directions,  reminding  one  of  witches'- 
brooms  in  trees.  Numerous  outposts  of  this  kind  give  a  peculiar  lumpy 
appearance  to  the  colony  as  seen  by  the  naked  eye.  This  peculiar  mode 
of  branching  was  clearly  to  be  seen  in  M26,  where  it  originated,  and  was 
frequent  throughout  subsequent  transfers.  Instead  of  single  threads 
reaching  out  in  this  way,  the  mycelium  sometimes  formed  fascicles  which 
would  grow  out  rapidly  into  new  territory  without  branching,  then  sud- 
denly branch  profusely,  forming  a  dense  brush.  These  two  rare  characters 
were  striking  in  effect  both  to  the  naked  eye  and  under  the  microscope. 
Nearly  every  transfer  from  M26  or  its  descendants  gave  colonies  with 
very  strikingly  marked  sectors,  characterized  essentially  by  abundant 
conidial  production,  and  therefore  dark  in  color.  The  other  sectors  bore 
few  conidia,  were  pale,  and  being  a  trifle  less  rapid  in  growth  they  were 
usually  crowded  out  (PI.  XXX,  lower  fig.).  These  characters  were  main- 
tained through  many  transfers.  (See  PI.  XXXI.) 

Variation  in  aerial  mycelium. — Saltant  sectors  and  their  progeny 
often  differed  from  the  originals  in  the  abundance  and  character  of  the 
aerial  mycelium.  In  some  cases  it  was  so  scant  as  to  be  unnoticeable; 
in  other  cases  so  abundant  and  floccose  as  to  obscure  from  vision  the 
colony  beneath.  In  character  it  varied  from  loose  and  fluffy  to  "ropy," 
the  latter  term  indicating  a  tendency  of  many  mycelial  strands  to  twine 
together  (Fig.  5,  c,  p.  104) .  In  other  cases  it  collected  in  clumps,  the  process 
being  attended  by  peculiar  distortions  (Fig.  6,  e) .  In  some  saltants  these 
clumps  were  abundant  and  aggregated;  in  others  few  and  scattered  (PI. 


144 

XXII,  XXIII).  The  occurrence  of  clumps  of  mycelium  upon  the  surface 
of  the  cultures  has  been  stressed  by  Ravn  (91)  as  of  taxonomic  importance 
(cf.  also  with  PL  XI,  XIII,  XXIII  (below),  XXVIII).  H.  No.  13,  in  one 
small  sector,  showed  eight  white  clumps;  the  balance  of  the  plate,  none.  In 
transfer  M71  the  clumping  character  seemed  lost,  but  the  following  transfers 
were  pale  in  type.  In  other  cases  the  clumping  habit  seemed  to  be  fixed 
and  characteristic  (PI.  XXVIII). 

Variability  in  colony  color. — The  color  is  mainly  due  to  abundance  or 
scarcity  of  conidia  or  to  abundance  or  lack  of  aerial  mycelium,  or  to  both. 
The  white  aerial  mycelium  is  practically  without  conidia.  Transfers 
from  sectors  with  much  white,  sterile  aerial  mycelium  were  not  always 
constant  in  these  characters,  but  in  many  instances  they  were  so;  for 
example,  M72,  derived  from  single  conidium  Cl-1,  and  M78,  derived  from 
M26.  Differences  quite  comparable  with  these  were  noted  by  Crabill  (36) . 

Zonation  was  well  marked  in  some  saltants  and  almost  entirely  lacking 
in  others  (PI.  IX,  3,  4,  5,  20).  Some  saltants  formed  sclerotia  abundantly 
though  the  originals  did  not  do  so.  Density  of  colony  also  differed,  some 
saltants  producing  colonies  of  much  denser  growth  than  others. 

Variability. — -Variability  itself  was  a  distinctive  character  in  certain 
instances.  Thus,  while  the  original  of  any  given  saltant  is  usually  fairly 
constant  in  its  characters  and  only  occasionally  gives  rise  to  saltants,  one 
saltant,  M26  (PI.  XXX,  below;  XXXI),  was  definitely  characterized  by 
the  fact  of  its  inconstancy  (see  page  143). 

Many  saltants  were  tested  as  to  their  infecting  power  and  their  rotting 
power,  but  no  real  difference  in  these  respects  was  noticeable  between  the 
different  saltants,  or  between  the  saltants  and  H.  No.  1 .  Since  many  species 
of  Helminthosporium  can  infect  many  cereals  this  power  may  be  rather 
fundamental  in  the  genus  and  thus  not  be  so  readily  subject  to  saltation  as 
are  less  fundamental  characters.  I  have  no  means  as  yet  to  measure  slight 
differences  in  either  virulence  or  rotting  power.  It  may  be  mentioned  here 
that  Ravn  (91)  states  that  culture  upon  dead  substrata  diminishes  the  vir- 
ulence of  Helminthosporium.  Whether  such  diminution  was  permanent 
or  merely  a  temporary  modification  he  did  not  determine.  Edgerton  (51) 
reports  that  different  races  of  Glomerella  differ  in  virulence. 

Correlation  of  characters  in  saltation. — Certain  correlations  of  characters 
are  noticeable;  thus,  colonies  of  slow  linear  growth  were  usually  high  in 


145 

conidial  production  and  rice  versa.     Correlations  observed  are   indicated 

as  follows: 

Slow  linear  growth* >high  conidia-production 

Much  aerial  mycelium >low  conidia-production 

Pale  colony* ^rapid  growth 

Thickening  of  conidia ^shortening  of  conidia 

Pale  colony >low  conidia-production 

Clumping  of  mycelium »low  conidia-production 

1  he  differences  in  colony-color  and  growth-rapidity  here  noted,  are 
much  like  those  described  by  Edgerton  (51)  in  Glomerella  plus  and  minus 
strains.  Crabill  (36)  notes  also  a  correlation  in  that  his  minus  strains  were 
always  of  more  rapid  growth  than  the  plus  strains. 

TENDENCIES  IN  SALTATION 

Saltants  showing  very  low  conidia-production,  verging  on  sterility, 
coupled  with  paleness  of  colony,  occurred  with  the  greatest  frequency.  A 
type  with  increased  conidia-production  and  of  slow  growth  was  next  in 
frequency.  The  latter  of  these  types  was  the  most  frequently  thrown  dur- 
ing the  early  period  of  my  work  though  it  has  been  rare  recently.  On  the 
other  hand,  the  former  type,  which  rarely  appeared  at  first,  is  now  the  most 
common.  A  type  characterized  by  thickness  of  conidium,  as  M6,  M8,  etc., 
has  been  frequent  all  the  time.  These  three  types  were  by  far  the  most 
common,  and  may  be  said  to  show  the  three  tendencies.  Markedly  short- 
conidia  saltants  were  few,  as  were  also  clump-bearing  types  that  possessed 
permanence.  Strains  that  threw  either  of  the  two  types  first  mentioned 
above  were  very  likely  to  continue  to  throw  similar  types.  The  same  may 
be  said  of  clump-bearing  types. 

STABILITY  OF  THE  SALTANTS 

Many  saltants  have  been  tested  in  various  ways  to  determine,  to  some 
degree,  their  constancy.  Through  numerous  transfers  on  corn-meal  agar 
the  O  colony  and  the  M  colony  of  many  saltants  have  been  carried  side  by 
side.  Under  such  conditions,  though  the  original  may  give  rise  to  new 
saltations  or  the  saltant  may  saltate  further,  the  main  portion  of  both  the 
O  and  M  colony,  as  a  rule,  maintains  its  characters. 

It  is  manifestly  impossible  to  test  all  the  saltants  to  ascertain  what 
their  future  behavior  will  be.  All  that  can  be  done  at  present  is  to  record 
certain  observations  concerning  them.  Several  saltants  possessing  strongly 
distinctive  characters  have  been  repeatedly  transferred  and  have  maintained 
their  characters  through  all  of  these  transfers ;  and  as  far  as  can  be  foreseen 


146 

are  as  stable  in  their  present  form  as  are  other  fungi.  Thus,  saltants  with 
short  conidia  (as  M35  and  M40)  and  saltants  with  broad  conidia  (M6  and 
M8)  have  been  cultured  and  graphs  of  conidia  repeatedly  made,  the  sal- 
tant  maintaining  its  character.  For  example,  a  determination_of_measure- 
ments  of  conidia  of  M35 — made  after  several  transfers  and  the  lapse  of 
some  weeks — gave  the  following  data: 

Mo-  CV 

17.31  ±  .25  2.51  ±  .17  14.50  ±  1.05 

Comparison  of  the  above  data  with  data  of  Graph  65,  Fig.  P,  shows 
that  this  saltant  not  only  remains  far  below  H.  No.  1  in  length  but  is  also 
constant.  It  is  particularly  to  be  noted  that  all  comparative  conidial  meas- 
urements were  made  under  standard  conditions.  Other  characters  ex- 
hibited by  saltants,  such  as  color,  zonation,  and  aerial  mycelium,  are  sim- 
ilarly permanent  when  strongly  marked.  Saltants  are,  however,  subject 
to  further  saltation  and  indeed  in  some  instances  are  exceptionally  liable 
to  it,  for  example,  M26.  Not  all  suspected  examples  of  saltation  afforded 
by  variant  sectors  proved  to  be  permanent  in  character,  and  some  lost  their 
distinguishing  marks  after  one  or  a  few  transfers.  Such  instability  was  not 
observed  in  cases  of  conidial  length  and  breadth,  or  of  pronounced  pale 
colony-color,  but  was  more  commonly  noted  in  cases  of  slight  differences  of 
aerial  mycelium,  slightly  pale  color  of  colony,  clumping,  etc.  While  all  cul- 
tures were  carried,  for  convenience,  on  corn-meal  agar,  and  their  differences 
were  observed  on  this  medium,  all  that  were  studied  critically  were  passed 
through  other  media — autoclaved  wheat-shoots  and  live-wheat — to  deter- 
mine whether  such  passage  would  alter  the  character  of  the  saltant.  The 
saltant  characters  were  apparent  on  other  media,  as  green-wheat  agar  or 
beef  agar,  though  the  general  colony-character  of  both  original  and  saltant 
was  changed  by  the  medium.  After  passage  through  these  conditions,  or 
through  wheat,  they  were  inoculated  under  standard  conditions  for  all 
graphic  comparisons.  There  is  no  evidence  of  alteration  of  the  characters 
of  the  saltants  by  such  procedure.  In  other  words,  the  saltation  is  not  a 
phenomenon  associated  with  the  medium  and  ended  when  the  fungus  gets 
back  to  its  normal  habitat. 

STABILITY  OF  THE  SALTANTS  THROUGH  THE  CONIDIA 

Dilution  platings  of  conidia  of  well-marked  saltants  gave  colonies  all 
alike  and  with  all  the  characters  of  the  saltant,  showing  permanence  of 
these  characters  through  the  conidia. 


147 

APPARENT  REVERSIONS 

In  several  instances  where  colony  color,  aerial  mycelium,  or  partial 
sterility  was  the  saltant  character,  small  sectors  of  the  colony  were  so 
changed  as  to  resemble  closely  the  originals,  and  as  far  as  tests  were  applied 
could  not  be  distinguished  from  them  (PI.  XXXII);  in  no  case,  however, 
where  true  saltant  character  was  proved  by  constancy  through  several  trans- 
fers did  the  whole  stock  revert;  what  appeared  as  reversion  was  limited  to 
occasional  sectors  of  the  colony,  and  in  no  case  did  such  change  occur  in 
the  entire  margin  of  a  colony. 

SUPPOSITITIOUS  CAUSES  OF  THE  VARIANT  SECTORS 

Several  alternative  suppositions  other  than  that  of  saltation  may  be 
briefly  discussed  as  possible  causes  of  the  variant  sectors.  The  mycelium 
at  a  certain  point  may  become  weakened,  or  die,  and  the  change  in  equilib- 
rium resulting  may  cause  the  variant  sector.  Spores  of  another  Helmin- 
thosporium  or  of  some  other  organism  may  fall  into  the  colony  from  the  air, 
and  the  variant  sector  may  represent  merely  a  contamination.  The  in- 
oculum used  on  a  plate  that  shows  saltation  may  have  consisted  of  more 
than  one  strain  or  elementary  species  of  Helminthosporium.  The  first 
supposition  is  open  only  to  crude  experimentation,  while  the  second,  if 
valid,  implies  a  wonderful  Helminthosporium-richness  in  the  air  of  my 
laboratory  as  well  as  very  faulty  technique.  Since  saltation  occurred  after 
the  fungus,  H.  No.  1,  had  been  transferred  many  times  by  lifting  a  small 
bit  of  agar  from  the  edge  of  a  colony,  the  presumptive  evidence  that  no 
mixture  then  existed  is  very  strong.  The  following  experiments  bearing 
on  these  suggestions  may,  however,  be  worth  recording. 

Wounding. — A  culture  of  H.  No.  1  on  corn-meal  agar  was  allowed  to 
grow  to  a  diameter  of  about  4  cm.  Then  by  means  of  a  hot  iridium  wire 
the  mycelium  was  killed  at  the  points  indicated  in  PI.  XXIX,  above.  In 
all  cases  the  uninjured  parts  soon  entirely  outgrew  the  wounds,  and  the 
whole  colony  presented  an  entire,  normal  outer  border  with  no  evidences  of 
saltation.  In  some  instances  a  clear  straight  line  extended  from  the  point 
of  wounding  nearly  to  the  edge  of  the  colony.  Evidently  disturbance 
of  equilibrium  such  as  this  did  not  cause  saltation. 

Mixed  planting. — Acting  on  the  'knowledge  that  the  saltants  were 
frequently  slow-growing,  and  thinking  that  possibly  ordinary  transfers 
might  be  mixtures  of  two  or  more  races,  of  which  the  slower-growing  one 
ordinarily  remained  masked,  M8,  a  well-characterized  saltant,  was  planted 


148 

on  a  corn-meal  agar  plate  and  allowed  24  hours  to  grow,  by  which  time  a 
vigorous  mycelium  had  developed.  A  goodly  quantity  of  conidia  of  H. 
No.  1  was  then  placed  in  the  midst  of  this  young  but  well-established  M8 
colony,  but  it  remained  uniform  to  full  occupation  of  the  plate^hpwing  no 
saltations. 

Implanting  conidia  of  H.  No.  1  in  a  partly  developed  colony  of  the  samz 
strain. — This  experiment  was  conducted  like  that  of  wounding  except  that 
instead  of  using  the  hot  wire  conidia  of  H.  No.  1  were  implanted  at  the 
points  indicated  in  PI.  XXIX,  below.  All  implants  within  the  colony 
grew  sparingly  and  resulted  in  small  clumps  1 — 2  mm.  in  diameter  and 
highly  sporiferous  (PI.  XXX,  above).  Implants  at  the  edge  grew  poorly, 
but  those  a  few  millimeters  outside  the  colony  became  established  and  grew 
well,  each  implant  developing  as  an  independent  colony  and  inhibiting  ad- 
vancement of  the  old  colony,  but  bearing  no  resemblance  to  a  saltant. 
In  one  case,  however,  such  implants  showed  marked  change  in  characters 
and  are  still  under  culture  as  saltants  (M70,  PI.  XXX,  upper  fig.),  though 
efforts  to  produce  other  saltants  in  this  manner  were  fruitless. 

Implanting  other  Helminthosporiums. — In  a  way  similar  to  that  of  the 
last  experiment  numerous  other  species  or  saltants  (e.g.  H.  No.  2  and  M6) 
were  implanted  in  an  H.  No.  1  colony,  and  always  with  the  result  that  the 
implant  either  failed  utterly  to  establish  itself  or  developed  as  an  entirely 
independent  colony  that  did  not  blend  with  the  main  colony,  being  in  this 
unlike  a  saltant  sector  in  character.  If  implants  were  put  about  3  mm.  be- 
yond the  tips  of  the  advancing  mycelium,  the  conidia  were  observed  to 
germinate  before  the  mycelium  of  the  H.  No.  1  colony  arrived,  but  even 
such  implants  became  entirely  submerged  and  lost. 

Two  entirely  distinct  types  of  Helminthosporium,  found  intermingled 
on  a  single  grain  of  wheat,  were  planted  together — an  oese  of  suspen- 
sion of  the  mixed  conidia — on  an  agar  plate.  The  resulting  colonies  gave 
the  two  types  of  Helminthosporium,  but  did  not  give  the  sectors  so  char- 
acteristic of  saltants. 

Saltation  not  due  to  parasites. — The  saltant  sectors  and  their  transfers 
often  differed  so  strikingly  from  their  originals,  particularly  when  they  bore 
few  conidia  and  had  much  white  aerial  mycelium  (see  PI.  XXVII) 
as  to  suggest  that  perhaps  the  great  difference  wras  due  to  a  parasite 
growing  in  the  Helminthosporium  colony.  Close  microscopic  inspection 
of  saltant  sectors  showed  that  there  was  only  one  type  of  mycelium  present, 
that  it  was  all  indistinguishable  from  Helminthosporium  mycelium,  and 


149 

that  no  conidia  indicating  contamination  were  present,  therefore,  if  the 
colony  were  parasitized  it  must  be  either  by  a  mycelium  like  that  of 
Helminthosporium  and  without  conidia,  or  by  some  virus  of  un- 
known character.  To  test  this  possibility  well-established  colonies  of 
H.  No.  1  were  inoculated  with  such  striking  saltants  as  M84.  Transfers 
of  M84  were  also  made  to  points  near  the  circumference  of  the  H.  No.  1 
colony.  If  M84  bore  a  parasite  of  any  kind  this  parasite  might  be  ex- 
pected to  invade  and  overgrow  the  H.  No.  1  colony.  This  it  did  not  do, 
but  the  two  colonies  halted  a  few  millimeters  apart  in  the  manner  char- 
acteristic of  two  Helminthosporium  colonies.  It  is  quite  clear  that  the 
idea  of  colony  parasitism  is  untenable  in  this  connection. 

Position  of  inoculum. — Since  it  was  possible  that  the  differing  appear- 
ances presented  by  the  various  sectors  might  be  due  to  the  position  of  the 
mycelial  strands  in  or  on  the  agar,  that  is,  on  top  of  it,  in  it,  or  below  it, 
tests  were  made  in  three  ways:  1,  by  placing  conidia  in  an  oese  of  w^ater  on 
the  surface  of  poured  agar;  2,  by  similarly  placing  conidia,  without  water, 
in  a  shallow  scratch  made  in  the  agar;  3,  by  so  cutting  the  agar  that  a  flap 
about  a  square  centimeter  could  be  lifted  and  inoculated  on  the  lower  side, 
that  is,  the  side  in  contact  with  the  glass,  the  flap  being  then  put  back  in 
place.  These  three  modes  of  inoculation  resulted  in  colonies  of  indistin- 
guishable character. 

SALTATIONS  FROM  SINGLE  CONIDIA 

Eight  separate  pure  cultures  were  made  from  single  conidia.  The 
eight  colonies  were  under  careful  microscopic  control  from  the  time  of 
planting  the  conidia,  through  germination,  and  until  the  colony  was  well 
developed,  and  it  is  certain  that  in  each  instance  the  colony  was  from  a 
single  conidium.  These  pure  strains,  all  alike  in  colony  character,  were 
labeled  Cl,  C2,  C3,  etc.  Well-marked  saltants  appeared  in  four  of  them 
as  follows: 

72 
Cl 

110 


119 
C2..  120 


108 
109 

121  

122 

128 

150 


68. 

.95 

114 

115 

116 

C3  

36 

117 

111 

112 

113  . 

128a 

106 

f 

126 

107  

..125  

} 

127 

C5.. 

37 

Thus,  it  will  be  seen  that  single  conidium  3  gave  rise  to  sixteen  clearly 
denned  saltants;  C5,  to  one;  Cl,  to  two;  and  C2,  to  seven — demonstrating 
absolutely  that  these  saltants  were  not  due  to  impurity  of  cultures.  Evi- 
dently the  saltant  sectors  do  not  result  from  contaminations. 

FREQUENCY  OF  SALTATION 

It  is  impossible  to  give  any  mathematically  accurate  statement  as  to 
the  frequency  of  saltation  in  Helminthosporium.  One  hundred  and 
twenty-six  variant  sectors  were  selected,  transferred,  and  more  or  less 
studied;  and  this  number  could  easily  have  been  doubled  or  trebled.  It 
is  not  probable  that  all  the  forms  in  these  sectors  were  truly  saltants; 
doubtless  some  of  them  were  mere  modifications,  but  the  number  that  were 
permanent  in  character  is  large.  How  many  of  these  saltants  agreed  with 
each  other  in  observable  characters  it  is  also  impossible  to  say,  but  since 
they  arose  independently  it  may  be  that  they  do  not  often  agree  absolutely. 
The  percentage  of  saltants,  based  on  those  theoretically  possible,  is,  how- 
ever, small  even  in  races  that  are  most  actively  saltating.  Thus  in  a  colony 
6  cm.  in  diameter  there  are  probably  more  than  5,000,000  cells,  and  theo- 
retically it  appears  probable  that  saltation  occurs  in  a  single  mycelial  cell, 
or  perhaps  by  the  union  of  two  cells,  yet  saltations  occur  with  even  less 
frequency  than  one  to  each  6-cm.  colony,  therefore  less  than  once  out  of 
5,000,000  possibilities.  In  this  connection,  though  no  direct  comparison 
is  possible,  it  may  be  noted  that  East  (47)  considers  the  occurrence  of  twelve 
inherent  variations  in  observations  made  on  100,000  hills  of  more  than  700 
varieties  of  potatoes,  that  is,  about  1:10,000,  as  an  unexpectedly  high  rate 


151 

of  frequency.  In  tobacco  only  one  bud  variant  was  noted  in  200,000 
plants.  Benedict  (16)  regards  the  production  of  50  new  Boston  ferns  in 
fifteen  years  as  rapid.  East  (46)  notes  that  all  of  the  asexual  variations 
have  been  losses  of  characters. 

The  pedigrees  of  the  various  Helminthosporium  saltants  which  I  have 
studied  are  indicated  in  the  following  table: 


PEDIGREE  TABLE  OF  SALTANTS 


65 

So     /  81 
59  \  82 

53*,  56*,  57*,  58 

3  

...26*....- 

60,61 

78 

89* 

f   52. 

74 

90* 

86 

96 

H.No.  7...-!   85 

4 

73 

76 

104 

103 

69 

f   98 

5* 

9*     \ 

92 

H.No.  8...^   99 

93 

I  100 

,   94 

:QQ 

f   69 

00 

88a.. 

88b 
88c 

H.No.9...<j   101 

(  102 

,   88d 

H  No  13     75 

13 

.  .  .71 

15*.... 

.  .  .64* 

f       *L  £ 

H.No.  1... 

32  

...79 

£.  r\ 

66 
H.No.14..-!   67 

35    .  A 

62 

I   91 

1 

63 

40  ..   .  < 

130  v 

77 

132 

(  131 

H.No.  17  129 
Unknown  27  . 

f  105 
....    84 
[   87 

1 

122 

134 

H.No.  34  97 

135 

49  

...80 

H.No.  39.  .  .Gave 
tants 

many  sal- 
not  num- 

f  118 

bered 

• 

83 

123 

(  124 

1*,  6,  7, 

8,  10,  12,  V 

t,  16-19*,  20,  21*-25, 

28-30*,  . 

51*,  32*,  33* 

,  34*,  38*,  39,  41-48, 

(  50,  51,  1 

0* 

*Numbers  followed  by  an  asterisk  are  pictorially  represented  in  the  plates. 


152 

The  apparent  paucity  of  saltation  in  the  strains  other  than  H.  No.  1 
may  be  due  in  large  part  to  the  fact  that  these  strains  have  been  cultured 
to  much  less  extent,  though  there  is  also  evidence  that  H.  No.  1  really  is 
more  actively  saltating  than  are  the  other  strains;  indeed  several  strains, 
as  H.  No  2  and  H.  No.  29,  have  given  no  evidence  of  sal tattoB-r- -Saltation 
seemed  to  be  as  frequent  in  cultures  derived  from  single  conidia  as  from 
other  cultures. 

SALTATIONS  OCCURRED  ON  VARIOUS  MEDIA 

Saltation  was  not  confined  to  corn-meal  agar,  but  was  seen  to  occur 
also  on  green-wheat  agar  and  on  washed  agar.  The  discrepancy  in  conidial 
measurements  on  two  shoots  (one  in  plate  e  and  one  in  e' ,  cited  on  p.  12i) 
may  have  been  due  to  saltation  on  the  washed  agar.  H.  No.  34  as  well 
as  H.  No.  1  showed  saltation  under  standard  conditions,  that  is,  on  washed 
agar  on  which  wheat  shoots  were  laid.  Several  of  the  shoots  bore  only 
sterile,  white  aerial  mycelium,  while  the  others  were  black  owing  to  the 
usual  number  of  conidia.  Repeated  transfers  demonstrated  the  perma- 
nence of  these  characters. 

SALTATIONS  AND  MODIFICATIONS  OCCURRING  IN 
TEST-TUBE   CULTURES 

Certain  cultures  received  from  correspondents  under  the  label  Hel- 
minthosporium  remained  largely  or  quite  devoid  of  conidia.  The  fol- 
lowing are  brief  descriptions  of  such  Helminthosporiums. 

H.  No.  11,  of  which  graph  of  conidial  length  is  given  in  Fig.  S  (cf. 
with  Graph  42,  Fig.  K)  and  conidial-breadth  in  Graph  101,  Fig.  V,  dif- 
fered in  general  colony-characters  from  H.  Nos.  1,  3,  etc.,  but  most  mark- 
edly in  that  it  remained  for  the  most  part  without  conidia.  Conidial 
septation  is  given  in  Fig.  T,  Graph  87. 

H.  No.  12,  which  was  received  under  the  label  "H.  gramineum  (?)" 
evidently  had  sometime  borne  conidia,  but  transfers  to  many  media  under 
many  conditions  gave  me  none  in  any  case. 

H.  No.  17,  also  labeled  H.  gramineum,  under  standard  conditions 
on  wheat  and  corn  usually  produced  mycelium  with  no  conidia,  though 
in  one  case  one  wheat  shoot  gave  conidia,  while  five  others  in  the  same 
dish  gave  none.  From  this  one  shoot  the  conidial-length  Graph  97  (Fig. 
U)  was  made. 

It  seems  to  me  that  the  three  cases  just  mentioned  should  be  regarded 
as  those  of  saltants  which  have  outstripped  their  originals  in  the  test- 
tube  conditions,  while  the  rare  cases  in  which  they  do  bear  conidia,  par- 


153 

ticularly  in  the  case  of  H.  No.  17,  given  above,  are  to  be  regarded  as  fur- 
ther saltation  or  as  reversions.  In  all  of  these  cases  of  sterile  cultures 
the  mycelium,  so  far  as  can  be  judged,  is  like  that  of  Helminthosporium, 
but  the  colony-characters  are  altered  in  ways  easily  compatible  with  the 
changes  following  loss  of  conidia-producing  power  and  consequent  change 
in  vegetative  vigor.  Ravn  (91)  mentions  frequent  sterility  of  cultures 
in  connection  with  Helminthosporium. 

H.  No.  18,  labeled  H.  avenae,  gave  conidia  only  once,  on  one  wheat 
shoot,  although  numerous  trials  were  made.  Its  case  is  almost  exactly 
like  that  of  H.  No.  17.  From  the  very  few  conidia,  though  inadequate, 
Graph  98  (Fig.  U)  was  made. 

H.  No.  19,  labeled  "H.  gramineum"  rarely  gave  conidia  under  any 
conditions,  though  somewhat  more  freely  than  either  of  the  two  preced- 
ing numbers.  (See  Fig.  U,  Graph  99.) 

H.  Nos.  13  and  14,  labeled  H.  sativum,  and  Nos.  15  and  16,  labeled 
H.  teres,  are  particularly  interesting  as  showing  variations  in  test-tube 
culture.  H.  No.  13  is  a  lineal  descendant  of  H.  No.  14  while  H.  No.  16 
is  a  similar  descendant  of  H.  No.  15.  Graphs  of  these  four  strains  also 
are  given  in  Fig.  U  (Nos.  93-96).  It  will  be  observed  here  that  the  differ- 
ences between  Nos.  15  and  16,  which  are  separate  cultures  from  the  same 
original  isolation,  are  greater  than  the  differences  between  strains  known 
to  be  quite  distinct.  Graphs  of  conidial  breadth  of  H.  Nos.  13,  14,  15, 
and  16  are  given  in  Fig.  V. 

H.  No.  20,  labeled  H.  teres,  appears  to  be  an  excellent  example  of 
saltation  in  test-tube  culture.  It  was  so  markedly  different  from  other 
cultures  that  during  the  several  months  in  which  I  was  ignorant  of  its 
origin  I  thought  that  here  was  a  clear  case  of  difference  between  the  Eu- 
ropean and  the  American  species.  Subsequent  word  from  Dr.  Wester- 
dijk  advised  me  of  the  American  origin  of  this  strain,  and  that  she  had 
received  it  in  1914  from  Bakke,  isolated  from  barley.  It  thus  seems  that 
this  culture  from  Dr.  Westerdijk  is  a  direct  descendant  of  the  culture  on 
which  Pammel,  King,  and  Bakke  based  their  description  (90),  and  that 
it  now  differs  markedly  from  that  description  as  well  as  from  a  culture 
(H.  No.  3)  which  I  received  from  Dr.  Bakke  which  was  also  taken  from 
the  same  plots  that  gave  the  original  culture  and  was  regarded  by  Dr. 
Bakke  as  being  identical  with  H.  sativum.  Among  my  notes  made  before 
I  knew  of  the  origin  of  the  culture  I  find  this  memorandum:  "H.  No.  20 
is  quite  distinct  from  all  other  forms  in  my  cultures  in  its  quite  uniformly 
6-septate  conidium  with  its  squarish  middle  cell.  Its  colony-characters 


154 

are  also  distinct."  (See  PL  IX,  No.  20.)  Conidial  length  is  given  in 
Fig.  U,  Graph  100;  breadth,  in  Fig.  V,  Graph  102;  septation,  in  Fig.  T, 
Graph  88.  (See  also  photomicrographs  of  conidia  in  PI.  XXVI,  d.) 
By  comparing  these  graphs  and  data  with  those  of  H.  No.  3  and  H.  No.  1 
it  will  be  seen  that  the  conidia  are  quite  short  and  a  trifle—thick.  The 
coefficient  of  cylindricity — 78  for  subcylindrical  conidia,  74  for  the  more 
elliptical  ones — is  the  highest  coefficient  shown  in  any  of  my  strains  (cf. 
with  page  119).  It  seems  very  probable, therefore,  that  either  the  culture 
was  contaminated  in  Dr.  Westerdijk's  laboratory  or  the  other  culture  in 
Dr.  Bakke's  hands;  or  that  Dr.  Bakke's  description  was  erroneous  or  that 
one  of  the  cultures  was  contaminated  by  me;  or  that  the  facts  represent 
a  real  hereditary  change  in  morphology  during  a  long  series  of  transfers — 
and  I  incline  strongly  to  the  last  of  these  alternatives. 

H.  No.  23  showed  what  appears  to  be  a  modification  rather  than  a 
saltation,  in  that  in  the  original  culture  received  from  Miss  Weniger  there 
were  many  abnormal  tri-pointed  conidia  (see  page  101).  It  is  suggested 
that  a  change  somewhat  like  this,  if  permanent,  may  have  given  rise  to 
the  forms  with  unequal  central  cell,  for  example,  to  H.  No.  29. 

All  of  the  above-mentioned  examples  appear  to  represent  clear-cut 
cases  of  change  in  morphological  character  in  test-tube  culture.  The 
only  essential  difference  between  these  changes  in  test-tube  culture  and 
the  saltation  reported  in  Petri  dishes  is  that  in  the  cases  of  the  Petri  dish 
selection  of  the  saltant  was  voluntary,  while  in  making  transfers  from 
tube  to  tube  the  selection  was  accidental. 

SALTATIONS  IN  NATURE 

That  changes  do  occur  under  my  culture  conditions  renders  it  highly 
probable  that  they  also  occur  in  nature — in  the  fields.  Thus  a  saltant 
strain  may  become  established  on  one  wheat  plant,  form  large  numbers 
of  conidia,  gain  foothold  in  a  region,  and  then  enlarge  this  foothold,  per- 
haps to  cover  large  areas.  That  one  strain  may  thus  outgrow  another 
has  been  shown  by  Crabill  (36)  and  is  evident  in  my  own  work  (PI.  XXX, 
XXXI,  lower  figs.).  The  fact  that  so  many  strains  of  Helminthosporium 
differing  slightly  but  distinctly  from  each  other,  yet  agreeing  closely  in  gen- 
eral, can  readily  be  isolated  from  cereals,  indicates  that  probably  this  also 
has  naturally  happened,  and  that  in  the  fields  we  have  today  large  numbers 
of  races  or  strains  of  closely  related  forms  derived  more  or  less  recently  from 
a  common  parent  stock.  To  test  this  hypothesis  experimentally,  in  field  or 
greenhouse,  by  inoculation  with  pure  cultures,  and  later  isolating  organ- 


155 

isms  to  search  for  differences,  does  not  seem  a  promising  line  of  research 
because  negative  evidence  would  be  valueless,  while  positive  evidence 
would  be  obtained  only  most  rarely,  even  though  saltation  is  very  com- 
mon. Since,  even  in  my  most  rapidly  saltating  strains,  changes  occur  in 
only  1  out  of  5,000,000  cells,  and  re-isolations  from  soils  would  give  col- 
onies from  single  conidia  only,  that  is,  from  a  small  group  of  cells,  the 
evidence  of  saltation  by  this  method  of  investigation  could  reasonably  be 
expected  only  once  in  several  thousand  platings. 

NOTES  CONCERNING  SELECTED  INDIVIDUAL  SALTANTS 

Unless  otherwise  noted  the  permanence  of  the  saltant  characters 
were  tested  by  repeated  transfers.  Colony-characters  were  determined 
on  corn-meal  agar;  measurements  of  conidia  and  other  conidial  characters, 
under  standard  conditions. 

Ml.  Origin  slightly  zonated  (PI.  XXIII,  1),  few  conidia.  Ml-1  grew 
faster  than  its  origin;  ratio,  6.5:8;  characters  maintained  through  several 
transfers. 

M6-1.  Much  like  Ml,  but  with  decided  difference  in  conidial  breadth 
(Graph  70,  Fig.  Q). 

M8.  Growth  slow;  conidia  few,  pale,  and  thick  (Fig.  Q) ;  septa 
few.  The  squarish  cells  very  striking;  differences  apparent  also  on  green- 
wheat  agar. 

M12-3.  Quite  distinct  in  septation  and  breadth.  As  to  conidial  length, 
see  Graph  121,  Fig.  Y,  and  data. 

Ml 7-3.  A  distinct  variant  in  thickness,  septation,  and  shape.  As  to 
conidial  length,  see  Graph  138,  Fig.  Y,  and  data. 

M26.     (See  page  143,  and  PL  XXX  and  XXXI.) 

M35.  Characterized  by  its  very  short  conidia  (see  page  141  and 
Fl.  XXVI,  c). 

M36.     Derived  from  a  single-conidium  culture;  conidia  thick. 

M38.  The  colony  had  much  aerial  mycelium  and  was  quite  white, 
though  a  shade  of  black  from  the  surface-agar  showed  through  ( PI.  XXVII) . 
It  continued  through  many  transfers  as  a  pale  form  with  scant  conidia. 

M53.  (PI.  XXX).  Very  different  from  its  origin,  being  covered  with 
much  loose,  white,  aerial  mycelium,  rendering  the  whole  colony  white  and 
flurry  in  appearance,  while  the  original  colony  was  neither  white  nor  fluffy. 

M54  and  M55.     These  also  were  two  white,  woolly  colonies. 

M56  (origin,  PI.  XXX)  and  M60.  These  were  from  a  dark  fast- 
growing  sector  of  M26  and  maintained  character. 


156 

M57  (PI.  XXX,  below) — M61  were  from  pale,  fluffy,  slow-growing 
sectors  of  M26.  M61  eventually  split  up  into  many  pale  and  dark  sectors. 

M62.     The  colony  grew  faster  than  its  origin,  and  bore  but  few  conidia. 

M64  (PI.  XXVII).  The  colony  was  entirely  white  and  fluffy  from 
much  aerial  mycelium.  It  originated  as  a  pale  sector  in^M-Hr-k 

M65  and  M68.  These  were  much  like  M62,  and  maintained  character 
through  many  transfers. 

M70.  This  arose  where  two  colonies  of  H.  No.  1  had  been  implanted 
just  outside  the  edge  of  a  colony  of  H.  No.  1  (PI.  XXX,  upper  fig.).  Both 
these  colonies  were  strikingly  different  from  the  original,  with  much  white 
and  fluffy  aerial  mycelium  Transfer  from  one  of  them  showed  the  char- 
acter permanent  through  several  transfers  (PI.  XXV),  but  whether  this 
saltation  was  actually  induced  by  the  implanting  or  whether  a  saltant  was 
unconsciously  selected  for  implanting  can  not  be  toid. 

M72.  Origin  a  most  striking,  fluffy,  white,  sterile  sector.  Characters 
transmitted. 

M75.  A  non-clumpy  sector  from  an  original  that  bore  many  clumps. 
Grew  faster  than  its  original. 

M78.  Very  loosely  growing,  stringy,  wrhite,  few  conidia.  Character 
maintained,  but  the  saltant  soon  threw  many  strongly  contrasting  light 
and  dark  sectors,  which,  however,  did  not  show  permanence  after  several 
transfers. 

M79.     Origin  like  M78,  but  character  soon  lost  in  transfers. 

M80.     Very  fluffy  and  white  and  rapid-growing. 

M81.  Had  a  narrow  (1  mm.)  pink  line  parallel  to  the  colony-edge  and 
about  5  mm.  from  it.  This  line  advanced,  remaining  narrow,  as  the  colony 
grew. 

M83.  Originated  on  green-wheat  agar  as  a  black  irregular  sector  of 
smooth  surface,  the  remainder  of  the  original  having  a  fluffy  surface. 

M85  and  M86.  TWTO  very  fluffy  white  sectors.  One  of  these  main- 
tained its  individual  character;  the  other  did  not. 

M87.  Whitish  in  its  origin,  and  in  succeeding  transfers  this  character 
was  intensified. 

M88.     Very  fluffy  and  white;  permanent  through  many  transfers. 

M88a.  A  pale  sector,  a  transfer  from  which  threw  numerous  variant 
sectors,  some  light,  some  dark. 

M92.  Originated  in  thin,  sterile  sectors  (strands)  which  produced 
sclerotia  at  their  outer  ends.  In  transfers  the  colony  bore  many  sclerotia. 

M93.     White  and  fluffy. 


157 

M94.     Very  pale  and  with  many  clumps. 

M95.     Rapid-growing,    pale,    with    few   conidia   and    many   clumps. 
Several  sectors  later  reverted  to  an  appearance  like  that  of  the  original. 
Ml 01 — Ml 05  were  all  fluffy,  with  white  aerial  mycelium. 


The  discussion  of  the  causal  fungus  of  foot-rot  has  so  far  been  based, 
for  simplicity,  upon  H.  No.  1.  Other  strains  of  a  Helminthosporium  of 
the  same  general  type  have  been  isolated  from  cases  of  foot-rot  from  Madi- 
son county  and  have  been  proved  capable  of  causing  foot-rot.  For  ex- 
ample, in  the  spring  of  1920  five  isolations  from  one  lot  of  material  were 
made.  These  I  designate  as  H.  No.  la,  H.  No.  16,  H.  No.  Ic,  H.  No.  Id, 
and  H.  No.  \e.  All  of  these,  in  colony  character  and  morphology,  agree 
closely  with  H.  No.  1,  but  H.  No.  la,  &,  c,  d,  have  graphs  of  conidial  length 
as  shown  in  Fig.  W,  wrhile  H.  No.  \e  differed  materially.  It  is  obvious  that 
the  first  four  may  be  considered  as  of  one  strain,  the  last,  e,  of  another 
strain,  both  strains  differing  somewhat  from  H.  No.  1.  The  conidial 
breadth  of  H.  No.  la  was  as  follows: 

f  M  o-  CV 

13          5.88  =*=  .07          0.39  ±  .05          6.79  ±  .89 

Conidial  septation  of  H.  No.  la,  H.  No.  ib,  and  H.  No.  If,  is  given  in 
Figure  X,  Graphs  111-113. 

GENERAL  DISCUSSION  OF  SALTATION 

The  existing  differences  in  definition  and  usage  of  the  term  mutation, 
as  also  our  very  limited  knowledge  of  cytological  conditions  in  the  genus 
Helminthosporium  and  our  ignorance  as  to  whether  it  has  sexual  stages, 
have  led  me  to  select  the  term  saltation  for  the  variations  here  discussed. 

The  term  mutant  is  defined  by  Dobell  (43),  following  Wolf  (127)  and 
Baur  (11),  as  follows:  "By  mutation,  accordingly,  I  mean  a  permanent 
change — however  small  it  may  be — which  takes  place  in  a  bacterium  and 
is  then  transmitted  to  subsequent  generations.  The  word  does  not  imply 
anything  concerning  the  magnitude  of  the  change,  its  suddenness,  or  the 
manner  of  its  acquisition.  The  term  denotes  a  change  in  genetic  consti- 
tution. All  other  changes  which  are  impermanent — depending  generally 
upon  changes  of  the  environment — and  not  hereditarily  fixed,  are  called 
modifications.  The  word  'mutation'  has  been  used  with  such  different 
meanings  by  so  many  bacteriologists  and  others,  that  the  foregoing  state- 
ment seems  called  for."  Brierley  (28)  defines  a  mutation  as  "a  genotypic 
change  in  a  pure  line";  and  Vaughan  (121),  as  "Those  changes  in  form  or 


158 

function  which  persist  through  one  or  more  generations  after  the  cause  of 
the  alteration  has  ceased  to  operate." 

Since  the  variations  herein  reported  occur  in  structures  purely  vege- 
tative and  result  from  no  intervening  sexual  act,  they  are in _kind  compar- 
able with  vegetative  variation  known  elsewhere — bud  variation,  etc. — 
with  the  exception  that  since  the  mycelium,  consisting  of  a  single  row  of 
cells,  is  the  seat  or  origin  of  the  variations  the  case  is  morphologically 
simpler  than  where  tissues  are  involved,  as  in  bud  variation.  De  Vries 
(122)  classes  bud  variations  in  general  with  mutations  in  that  they  appear 
as  clear-cut  discontinuous  variations.  Many  examples  of  vegetative 
variation  have  been  studied  extensively  and  reported  upon  under  the 
terms  mutation,  saltation,  sporting,  etc.  Cramer  (37)  gives  a  very  com- 
plete summary  of  the  known  cases  in  1907.  East  (46),  Stout  (120),  Ghys 
(61)  and  Shamel,  Scott,  and  Pomeroy  (102,  103)  give  reports,  with  ex- 
tended bibliographies,  concerning  bud  variation  in  the  potato,  Coleus, 
chrysanthemum,  and  lemon  among  the  phanerogams.  The  transmission 
of  such  variations  in  the  potato  has  been  carefully  studied  by  East  (47, 
48,  49)  with  the  general  conclusion  that  these  asexually  appearing  varia- 
tions concern  characters  that  Mendelize  in  sexual  reproduction.  In  the 
Pteridophytes,  Benedict  (16)  reports  saltation  in  the  .Boston  fern.  Dobell 
(43)  summarizes  the  evidence  from  some  twenty-eight  papers  concern- 
ing variability  among  the  bacteria,  discussing  them  in  two  categories:  (1) 
physiological  mutations,  that  is,  changes  in  power  to  produce  ferments 
or  pigments;  and  (2)  morphological  mutations.  He  closes  the  first  part 
of  his  discussion  as  follows:  "It  seems  legitimate  to  conclude  from  the 
foregoing  facts  that  some  races  of  bacteria  are  able  permanently  to  acquire 
new  characters  under  certain  conditions."  He  considers  only  two  cases 
of  morphological  mutation,  citing  the  work  of  Barber  (7),  who  produced 
a  race  of  long  bacteria  by  single-cell  selection,  and  of  Revis  (94),  who 
claims  to  have  produced  a  new  race  of  Bacillus  coli  by  the  use  of  malachite 
green.  Both  Laurent  (76)  and  Le  Poutre  (77)  conclude  that  by  passage 
a  harmless  bacterial  species  may  acquire  real  virulence.  An  extensive 
resume  of  the  question  of  mutation  in  ,the  bacteria  is  also  given  by  Baerth- 
lein  (5).  Numerous  variations  of  yeasts,  both  morphological  and  phys- 
iological, are  reported  by  Guilliermond  (62). 

The  validity  of  the  conclusion  maintaining  that  there  is  mutation 
among  the  bacteria  and  yeasts  has  been  attacked  by  Brierley  (28)  on  the 
ground  that  the  changes  reported  as  mutations  are  merely  due  to  segre- 
gation of  organisms  of  aberrant  type  from  an  originally  mixed  popula- 


159 

tion.  The  very  large  mass  of  corroborating  positive  evidence,  though 
really  conclusive  only  when  based  on  single-organism  cultures,  makes  it 
extremely  probable,  to  say ,  the  least,  that  such  saltations  or  mutations 
do  occur  in  these  groups. 

Among  the  Eumycetes  several  examples  occur  of  what  appears  to 
be  saltation.  Edgerton  (50)  in  1908,  writing  of  Glomerella  rufomaculans, 
states  that  "of  the  more  than  thirty  collections  studied  from  over  twenty 
hosts,  with  less  than  a  half-dozen  exceptions  all  gave  at  least  slightly 
different  characters.  Even  the  two  collections  on  apples  from  Missouri 
and  Illinois  did  not  give  exactly  the  same  characters,  but  the  differences 
were  slight.  The  two  collections  from  apples  in  the  north,  however,  gave 
entirely  distinct  characters  from  the  more  southern  forms  on  the  same 
host.  The  southern  form,  especially  on  sugar  medium,  was  characterized 
by  very  rapid  growth  and  a  very  dark  greenish-black  color  of  the  sub- 
stratum and  aerial  hyphae;  while  the  northern  form  grew  more  slowly 
and  had  very  little  dark  color.  Generally  in  the  latter  the  aerial  hyphae 
were  colored  pink  from  the  profuse  development  of  conidia.  Even  the 
form  on  quince  collected  in  New  York  did  not  give  the  same  characters 
as  the  northern  form  on  apple.  The  forms  on  orchid,  Coffea,  and  Sar- 
racenia,  collected  in  the  same  greenhouse  at  the  same  time,  were  not 
exactly  alike  in  culture  media." 

Other  examples  were  given  by  Edgerton  (50)  in  1908  of  what  appears 
to  be  the  same  phenomenon  as  that  under  discussion  here,  but  as  there 
is  no  evidence  that  he  worked  with  single-conidia  cultures*  his  apparent 
variations  may  have  been  due — though  it  is  highly  improbable — to  segre- 
gation of  elementary  species.  His  general  conclusion  follows:  "The  only 
explanation  of  the  phenomenon  is  that  one  or  more  individuals  of  the 
original  form  changed  quite  suddenly  their  course  of  development  under 
cultural  conditions.  It  is  undoubtedly  a  Gloeosporium  of  the  Glomerella 
type,  with  the  development  of  the  perithecia  considerably  different  from 
other  known  forms.  Mutations,  so  far  as  is  known  by  the  writer,  have 
not  previously  been  recorded  among  fungi,  but  the  form  just  described 
seems  to  be  one  without  question." 

Here,  too,  should  be  mentioned  the  plus  and  minus  strains  of  Glom- 
erella studied  and  reported  on  by  Edgerton  (51)  from  single-conidia  cul- 
tures, though  this  may  represent  a  differentiation  of  sexes  rather  than  of 
races.  He  gives  several  citations  which  indicate  that  other  workers  have 


*In  a  personal  letter  dated  February  7,  1921,  he  writes  regarding  this  as  follows:     "All  of  the  cultures 
that  I  used  in  that  work  were  obtained  by  the  dilution  plate  method  and  presumably  came  from  single  spores." 


160 

studied  these  strains,  and  records  the  belief  that  the  culture  mentioned 
above  "as  a  possible  mutation  ....  was  really  the  minus  strain 
of  the  bitter-rot  fungus." 

In  1909  I  reported  with  Dr.  Hall  (Stevens  and  Hall,  118)  for  the 
genus  Ascochyta  variant  sectors  in  Petri-dish  cultures  quite~like  those 
reported  in  this  paper;  but  since  the  study  was  not  made  from  single- 
conidium  isolations  it  is  possible,  though  not  probable,  that  I  had  merely 
a  segregation  of  elementary  species. 

Shear  and  Wood  (105)  in  1913  reported  that  in  cultures  of  Glomerella 
started  from  a  single  ascospore  "an  important  variation  or  mutation  sud- 
denly occurred  in  the  fourth  generation  and  was  transmitted  through 
three  following  generations."  They  cite  other  variations,  less  permanent, 
which  they  regard  as  fluctuations. 

Burgeff  (31)  in  1914  reported  results  from  an  extensive  study  of 
asexual  variation  in  the  genus  Fhycomyces.  Working  from  single-spore 
isolations  he  got  great  diversity  in  many  characters. 

Crabill  (36)  in  1915  described  two  strains  of  Coniothyrium  pirinum 
which  he  designated  as  plus  and  minus  strains  that  differ  markedly  in 
several  characters,  particularly  in  size  and  abundance  of  pycnidia  (verg- 
ing on  complete  sterility),  and  in  color  of  colony.  He  says: 

"The  cultural  studies  show  that  minus  strains  may  arise  from  plus 
strains  by  a  sudden  sporting  or  mutation.  An  objection  might  be  raised 
that  these  cultures  were  impure,  i.  e.,  mixtures  of  two  strains.  In  antici- 
pation of  such  an  idea  it  seems  desirable  to  state  that  frequent  pourings 
of  dilution  cultures  were  used  to  preclude  such  a  possibility.  Progeny 
were  then  selected  only  from  well-isolated  plant,  microscopic  examination 

of  which  showed  that  each  was  derived  from  a  single  spore 

Both  strains  have  repeatedly  arisen  from  the  progeny  of  a  single  plus 
spore.  When  once  purified  the  minus  strain  remains  constant  from  gen- 
eration to  generation.  The  variation  is  apparently  occurring  in  only 

one  direction The  only  explanation  which  remains  is  that 

the  minus  strain  is  a  sport  or  mutant  arising  from  the  plus  strain  at  irreg- 
ular and  unprognosticable  intervals." 

The  plus  strain  by  sudden  sporting  gives  rise  to  the  minus  strain,  but 
minus  strains  were  not  seen  to  give  rise  to  plus  strains,  that  is  to  say,  the 
saltation  is  orthogenetic.  He  states  also  that  the  variation  apparently 
occurs  in  the  spore  and  not  in  the  mycelium,  which  is  quite  the  contrary 
to  my  findings.  He  finds,  in  agreement  with  my  work,  also  with  Kleb's 
law  (74),  that  the  minus  strain,  that  is  the  sporiferous  one,  grows  faster 


161 

than  the  plus  strain.  The  sectoring  and  the  colony  differences  which 
he  pictures  are  much  like  those  shown  for  Helminthosporium  in  this  paper. 
He  also  adduces  evidence  to  show  that  these  plus  and  minus  strains  occur 
in  nature  and  have  been  isolated  by  independent  workers.  He  attempted 
in  four  ways  to  induce  saltation  artificially  but  met  with  no  success. 
Crabill  (35)  has  reported,  in  abstract,  "a  somewhat  similar  mutation 
in  a  fungus  belonging  apparently  to  the  genus  Phyllosticta."  Blakeslee 
(21)  reports:  ".  .  .in  1912-13  I  found  numerous  variants  of  various  de- 
grees of  distinctness  in  the  offspring  of  a  single  plant  (Mucor)  obtained 
by  sowing  non-sexual  spores."  Writing  of  Mucor  genevensis  he  says  (20): 
"In  all,  somewhat  over  38,000  colonies  from  individual  sporahgiophores 
have  been  inspected  and  a  relatively  large  number  of  variants  of  different 
degrees  of  distinctions  have  been  obtained  ....  the  mutants 
tend  eventually  to  revert  to  the  normal  type.  Two,  however,  have  seemed 
more  stable."  He  concludes:  "They  add  to  the  evidence,  already  obtained 
from  other  groups,  that  mutations  are  not  restricted  to  processes  involved 
in  sexual  reproduction." 

Brierley  (27,  28)  reported  an  albino  Botrytis  cinerea  which  was  a  form 
with  pale  sclerotia  though  the  parent  form  always  had  black  sclerotia. 
This  albino  was  observed  to  arise  from  a  colony  derived  from  a  single  conid- 
ium  and  from  a  race  that  had  been  under  culture  for  considerable  time, 
always  producing  black  sclerotia.  The  purity  of  his  culture  seems  to  have 
been  carefully  guarded,  and  this  case,  though  standing  alone,  would  furnish 
positive  evidence  of  the  sudden  occurrence  of  a  hereditary  difference  in 
this  fungus. 

Dastur  (40)  in  1920  described  saltations  in  Gloeosporium  piperatum 
consisting  in  the  absence  or  presence  of  perithecia,  acervuli,  or  setae,  and 
in  the  development  of  aerial  mycelium.  He  says:  "Thus  all  of  a  sudden 
the  original  sterile  culture  broke  up  into  two  different  strains,  one  producing 
only  perithecia  on  sterilized  chilli  stems  and  the  other  forming  acervuli 
with  and  without  setae."  Some  of  these  strains  were  not  constant  in 
character,  but  others  persisted  through  many  transfers.  He  states  that 
great  or  sudden  variations  have  never  been  observed  from  conidial 
strains,  but  that  "in  cultures  made  from  perithecia  of  the  strain 
of  Gloeosporium  piperatum  incredibly  large  and  often  very  sudden  variations 
have  been  obtained."  Burger  (32)  in  1921  reported  mutation  of  several 
types  in  Colletotrichum,  involving  permanent  changes  in  many  characters. 
He  found  these  occurring  in  cultures  derived  from  single  spores  and  showed 
that  they  were  permanent  through  the  spores.  Jennings  (70),  who  worked 


162 

with  the  shelled  rhizopod  Difflugia,  states  that  most  of  the  work  on  uni- 
parental  reproduction  has  yielded  the  result  that  during  such  reproduction 
the  hereditary  constitution  (genotype)  appears  not  to  change  though  the 
organism  may  differ  much  in  outward  character.  Many  papers  cited 
support  this  view  though  some  are  opposed.  Jennings  sayeTwiIirregard  to 
his  own  results:  "After  many  generations  of  descent  from  a  single  progenitor, 
such  a  single  family  ....  has  differentiated  into  many  he- 
reditarily diverse  stocks."  These  diverse  stocks  differ  hereditarily  not  only 
with  respect  to  particular  single  characters  but  also  with  respect  to  the 
combination  of  characters.  Many  individuals  of  uniparental  reproduc- 
tion have  shown  a  marked  permanence  of  hereditary  character  in  single 
lines  of  descent,  all  the  progeny  being  like  the  parent  in  hereditary  consti- 
tution; and  further,  many  such  lines,  diverse  in  hereditary  constitution  may 
exist  in  a  population,  and  the  effects  of  selection  consist  mainly,  if  not 
entirely,  in  the  isolation  of  such  diverse  lines. 

East  sees  no  reason  to  believe  bud  variation  different  from  germinal 
mutation  and  says  that  it  may  be  progressive,  digressive,  or  retrogressive. 
Bateson  (10)  holds  that  bud  variations  are  due  to  qualitative  cell-division 
in  somatic  tissues,  giving  somatic  segregation  of  unit  factors.  East  points 
out  that  in  the  large  majority  of  cases  of  bud  variation  there  has  been 
simply  the  loss  of  a  dominant  character  and  hence  the  appearance  of  a  re- 
lated recessive  character.  In  some  cases  there  is  absolute  disappearance 
of  the  dominant  character;  in  other  cases  it  appears  to  be  latent,  and  it  may 
reappear.  Variations  in  color  constitute  over  70%  of  all  bud  variation. 
Colony  color  in  Helminthosporium  is  also  a  very  common  variable  but 
this  is,  in  all  probability,  entirely  incomparable  with  color  variation  in 
flowering  plants. 

Brierley  (28)  holds,  for  Botrytis,  that  even  if  sexuality  occurs,  the 
fungus  is  "on  all  evidential  criteria,  an  asexual  homozygotic  organism  in 
which  the  isolation  of  a  single  spore  strain  necessarily  implies  the  isolation 

of  a  'pure  line'  " A  genotypic  change  in  a  pure  line  is  a 

mutation."  Similarly,  Shear  and  Wood  (105)  regard  individuals  orig- 
inating from  single  spores  of  Glomerella  as  homozygous,  though  on  reason- 
ing differing  somewhat  from  that  of  Brierley.  Crabill  (36)  also  holds  that 
since  his  fungus  "reproduces  asexually  segregation  from  heterozygous 
parents  cannot  explain  the  origin  of  the  strains."  Accepting  Brierley 's 
criteria,  my  Helminthosporium  single-spore  isolations  are  equally  homo- 
zygotic. In  at  least  three  forms  on  cereals  Helminthosporium  is  known 
to  be  the  conidial  stage  of  the  ascigerous  genus  Pleospora.  As  ascigerous 


163 

stages  are  known  in  many  cases  to  arise  sexually,  it  may  be  expected  that 
all  perithecia  represent  sexual  stages  and  a  sexual  act.  The  particular 
forms  with  which  I  am  working,  that  is  H.  No.  1  and  derivatives,  have 
given  no  evidence  of  perithecial  formation  nor  is  it  actually  known  that 
they  possess  sexual  stages.  The  presumption,  however,  is  that  they  do, 
so  it  is  quite  possible  that  my  culture  of  H.  No.  1  is  derived  from  an  asco- 
spore,  that  is,  may  be  the  result  of  sexual  parentage;  and  this  sexual  act 
may  have  occurred  in  the  not  distant  past.  This  is  all  hypothetical  but  it 
appears  to  me  to  point  to  a  possibility  of  an  heterozygotic  condition  in 
Helminthosporium,  as  well  as  in  Glomerella  as  reported  by  Shear  and 
Wood  (105)  and  in  Coniothyrium  as  reported  by  Crabill  (35),  since  so  few 
generations  may  have  elapsed  since  fertilization  that  the  heterozygosis 
has  not  yet  been  eliminated.  Such  supposition  in  the  case  of  Botrytis  is 
less  tenable. 

It  is  suggestive  to  note  here  that  Dastur  (40)  found  variation  a  com- 
mon phenomenon  only  in  strains  that  were  recently  derived  from  perithecia ; 
also  that  bud  variation  is  more  common  in  hybrids  (East,  46).  It  is  there- 
fore thinkable  that  such  of  my  strains  of  Helminthosporium  as  are  saltating 
are  of  recent  ascigerous  origin,  while  others  that  are  not  saltating  (for 
example  H.  ravenelii  and  H.  geniculatum]  are  of  distant  ascigerous  origin. 

If  heterozygosis  be  eliminated  from  the  discussion  two  other  possible 
explanations,  suggested  by  Brierley  regarding  Botrytis,  may  be  considered 
here,  namely,  that  of  nuclear  transference  during  mycelial  anastomosis  (Fig. 
5,  p.  104)  or  that  of  cytoplasmic  contamination  by  such  anastomosis.  Evi- 
dence on  these  questions,  both  from  cytology  of  the  mycelium  and  from 
knowledge  of  sexuality,  is  quite  lacking.  Accepting  none  of  the  above 
hypotheses,  the  saltation  would  be  a  mutation  in  the  strictest  sense  of  the 
term. 

Reported  mutations  in  Aspergillus  and  Penicillium  described  by 
Arcichoyskij  (2),  Waterman  (125),  and  Schiemann  (100),  and  said  to  be  in- 
duced by  environmental  changes,  are  quite  extensively  discussed  by 
Brierley  (28),  who,  repeating  much  of  their  work,  concludes  that  when 
the  fungi  showing  these  changes  are  returned  to  their  original  environ- 
ment they  resume  their  original  aspect;  that  in  fact  the  changes  were 
mere  modifications  due  to  environment. 

The  cases  reported  by  Brierley,  Burgeff,  Blakeslee,  and  Crabill,  and 
my  own  work  reported  herein,  all  based  on  single-spore  culture  and  car- 
ried under  observation  for  sufficient  time  to  give  assurance  of  permanence, 
constitute  complete  proof  of  the  occurrence  of  the  phenomenon  of  sud- 


164 

den  change  in  character  among  the  fungi.  The  evidence  from  Edgerton, 
Shear  and  Wood,  and  Dastur,  while  not  so  complete,  is  strong  collaterally. 
It  would  seem  from  all  this  evidence  that  this  phenomenon  is  common 
and  widely  distributed  among  the  fungi,  though  unquestionably  it  is  more 
common  in  some  species  and  races  than  in  others. 

TAXONOMY 

The  classification  of  these  Helminthosporium  "forms,"  indeed  of  all 
the  fungi  imperfecti,  presents  unusual  difficulties.  That  they  are  only 
"forms"  of  which  we  do  not  yet  know  the  ' 'perfect"  stage,  is  no  more 
relief  from  the  necessity  of  classification  than  is  incomplete  knowledge 
adequate  reason  for  delay  in  attempts  to  classify  other  plants.  In  the 
present  instance  some  well-defined  "species,"  in  the  old  sense  of  the  word, 
stand  out — for  example  H.  ravenelii — while,  on  the  other  hand,  several 
of  the  strains  of  Helminthosporium  in  my  collection  differ  in  one  or  more 
slight  ways  yet  agree  with  each  other  closely  in  general  type.  For  ex- 
ample, my  H.  No.  1,  H.  No.  11  (isolated  by  Stakman  from  wheat  in  Min- 
nesota), H.  No.  13,  isolated  by  Durrell  in  Iowa,  H.  No.  23,  isolated  by 
Weniger  in  North  Dakota,  and  an  unnumbered  one  isolated  by  Hoffer 
in  Indiana,  are  all  clearly  the  same  general  type  of  organism,  and  yet 
they  differ  from  each  other  in  minor  particulars. 

It  is  evident  that  we  have  in  the  genus  Helminthosporium  large 
numbers  of  races  that  vary  consistently  and  constantly,  though  but  slightly, 
from  each  other.  These  variations  may  be  morphological  in  the  usual 
sense  of  the  term,  or  as  shown  in  cultures,  or  as  demonstrated  biomet- 
rically.  It  is  quite  probable  that  here,  too,  there  are,  as  elsewhere  in  the 
fungi,  differences  in  virulence,  and  therefore  in  biologic  relationship,  and 
physiologically.  Examples  are  numerous  among  the  fungi  where  such 
comparatively  minor  differences  are  regarded  as  of  specific  rank  and  the 
new  group  is  designated  by  a  new  binomial.  There  are  also  numerous 
examples  where  such  slightly  variant  types  are  regarded  as  varieties  or 
races  0f  the  species.  These  varieties  or  races  have  been  variously  desig- 
nated as  follows  (or  by  the  equivalents  of  these  terms  in  other  languages) : 

Physiological  species  (Hitchcock  and  Carleton — 67) 

Species  sorores  (Schroeter — 101) 

Biologische  Spezies  (Klebahn — 73) 

Biologiske  Arten  (Rostrup — 95) 

Schwester  Arten  (Schroeter) 

Biologische  Rassen  (Rostrup— 95,  96) 

Specialisirte  Formen  (Eriksson) 


165 

Formae  speciales  (Eriksson — 55) 

Gewohnheitsrassen  (Magnus — 80,  81) 

Races  specialiees  (Marchal) 

Mikrospecies 

Biotypes 

Elementary  species  (de  Vries) 

Pure  lines  (Johannsen) 

Biological  forms 

Biological  races 

Fischer  (56)  adopts  the  practice  of  recognizing  as  distinct  all  forms 
which  differ  in  their  choice  of  hosts  in  so  far  as  the  hosts  belong  to  dif- 
ferent genera;  a  procedure  that  leaves  the  specific  rank  and  name  of  the 
parasite  subject  to  vicissitudes  arising  from  subsequent  changes  in  the 
conception  of  the  taxonomy  of  the  host.  It  is  yearly  becoming  more 
evident  that  distinctions  such  as  these  are  common  in  the  fungi  within 
what  were  previously  regarded  as  groups  of  specific  rank. 

Biologic  specialization  in  the  rusts  was  announced  in  1894  by  Eriks- 
son (55),  and  has  since  been  abundantly  attested  by  Stakman  (109), 
Stakman  and  Piemeisel  (111),  Stakman,  Piemeisel,  and  Levine  (112),  by 
Arthur  (3,  4),  and  by  others  (57,  68).  Abundant  evidence  that  it  occurs 
in  the  powdery  mildews  is  afforded  by  Neger  ( 85) , Salmon  (98) ,  and  Reed  (92) . 

The  first  demonstrated  cases  in  the  fungi  imperfecti  were  probably 
in  Helminthosporium,  reported  by  Ravn  (91).  It  was  demonstrated  in 
Septoria  by  Beach  (12).  Reed  (93),  summarizing  regarding  biologic 
specialization,  cites  papers  to  show  its  occurrence  in  the  following  genera: 
Synchytrium,  Albugo,  Peronospora,  Taphrina,  Claviceps,  Dibotryon, 
Rhytisma,  and  Colletotrichum. 

Evidence  that  there  is  differentiation  morphologically,  slight  but 
measurable  and  constant,  has  been  found  among  the  rusts  by  Arthur 
(3,  4)  who,  writing  of  Uromyces  on  Spartina,  says  that  "the  four  races 
of  this  species  exhibit  not  only  physiological  specialization  but  a  certain 
amount  of  morpholcgical  differentiation."  Similar  findirgs  are  reported 
by  Bisby  (17)  concerning  Puccinia  epilobii-tetragoni,  by  Stakman  and 
Fiemeisel  (111)  regarding  Puccinia  graminis,  and  by  Arthur  (3,  4)  regard- 
ing Dicaeoma  poculijormis  on  Phleum.  Brierley  (26)  has  demonstrated 
by  single-spore  cultures  the  existence  of  elementary  species,  morphologic- 
ally distinct,  writhin  the  species  of  Botrytis,  Penicillium,  and  Stysanus. 
Gaumann  (60)  has  shown  Peronospora  parasitica  to  consist  of  very  numer- 
ous races  separable  on  both  biologic  and  morphologic  grounds.  Similar 
findings  regarding  Plasmopara  are  reported  by  Wartenweiler  (124).  Pes- 


166 

talozzia  guepini  was  reported  by  Bartlett  and  La  Rue  ( unpublished  paper) 
to  consist  of  numerous  distinct  strains.  Ascochyta  chrysanthemi  was 
shown  to  consist  of  at  least  two  distinct  strains  by  Stevens  and  Hall  (118). 
Crabill  (34)  states  that  there  are  four  distinctly  different  types^  of  Phyl- 
losticta  pirina,  and  that  Coniothyrium  pirinum  (34)  is  similarly  composed 
of  distinct  races.  Burger  (32)  demonstrates  a  similar  condition  in  the 
genus  Colletotrichum.  Wiedemann  (126)  has  published  species  of  Peni- 
cillium  based  on  differences  shown  on  culture  media — a  procedure  very 
common  in  dealing  with  the  bacteria.  The  wide-spread  occurrence  of 
slightly  but  constantly  differing  varieties  within  the  species  of  fungi  is 
apparent;  also  that  two  diametrically  opposed  methods  of  procedure  are 
in  vogue  to  meet  the  situation.  One  method  gives  specific  rank  to  each 
elementary  species,  or  race,  or  strain;  the  other  restricts  binomial  desig- 
nation to  the  larger  groups  (the  collective  species),  recognizing  that  the 
latter  consist  of  numerous  smaller  groups — the  elementary  species  (or 
races  or  strains).  Lotsy  (79)  suggests  that  the  terms  "Linneon" — defined 
as  "a  total  of  individuals  which  resemble  one  another  more  than  they  do 
any  other  individuals" — and  the  term  "Jordanon" — for  the  elementary 
species — be  employed.  This  suggestion,  in  that  it  recognizes  the  ele- 
mentary species  as  properly  in  one  category,  and  the  group  of  elementary 
species  as  belonging  in  a  larger  category,  both  subordinate  to  the  genus, 
is  in  accord  with  the  general  discussion  of  " Aspects  of  the  Species  Ques- 
tion" (29)— see  particularly  conclusions  of  Britton  and  remarks  by  Coul- 
ter. The  difficulties  regarding  elementary  species  that  beset  the  taxono- 
mist  in  dealing  with  the  flowering  plants  are  manifoldly  increased  when 
the  classification  of  the  fungi  imperfecti  is  in  question.  Thus  in  the 
genus  Septoria  there  are  more  than  1200  named  species  usually  delimited 
from  each  other  by  barely  three  or  four  characters,  and  these  extremely 
variable.  Many  other  genera  present  conditions  equally  bewildering. 
The  result  is  that  it  is  absolutely  impossible,  even  with  the  type  speci- 
mens in  hand  (and  they  are  usually  unobtainable),  to  determine  species 
accurately.  It  is  highly  probable  that  many  of  the  forms  now  listed  as 
species  in  the  fungi  imperfecti  are  either  identical  or  merely  biologic  races 
— that  is  elementary  species.  To  designate  each  elementary  species 
either  in  the  fungi  imperfecti  or  elsewhere  by  a  binomial  defeats  the  very 
purpose  of  the  name  and  renders  it  not  only  useless  but  cumbersome. 
The  conceptions  of  Lotsy  and  of  Britton  and  Coulter  as  noted  above, 
seem  particularly  applicable  here  and  indicate  the  advisability  of  using 
a  binomial  to  designate  a  group  which  shall  comprise  many  elementary 


167 

species — a  course  that  I  have  already  followed  in  the  case  of  Colletotri- 
chum  (115)  and  which  was  followed  by  Elliott  (53)  in  dealing  with  Al- 
ternaria. 

My  H.  No.  1,  H.  No.  la,  H.  No.  16,  H.  No.  Ic,  and  H.  No.  Id,  the  cause 
of  foot-rot  in  Madison  county,  111.,  as  well  as  my  Helminthosporium  num- 
bers 3-9,  11-19,  22-27,  34,  37,  38,  42,  and  43,  all  belong  to  the  same  general 
type  and  are  characterized  by  a  conidium  that  tapers  toward  each  end  from 
a  point  of  greatest  thickness  which  is  nearer  to  the  base  than  to  the  apex 
of  the  conidium.  The  conidia  are  therefore  not  typically  cylindrical  or 
even  subcylindrical.  While  all  of  these  numbers  agree  in  general  type, 
many  of  them  differ  somewhat  from  others  in  the  collection.  Thus  Nos.  1 
and  3  differ  as  is  shown  in  Plates  IX,  XI -XII I,  and,  so  far  as  observed, 
in  this  character  only,  and  only  under  the  conditions  described.  Others 
differ  in  modal  spore-length  or  septation,  in  distinctness  of  zonation,  or  in 
other  minor  colony-characters  (PI.  IX).  Number  13  differs  slightly  even 
from  No.  14,  though  both  are  derivatives  from  the  same  original  culture; 
and  the  same  may  be  said  of  Nos.  15  and  16.  These  differences  which  now 
actually  exist,  are  probably  due  to  unconscious  selection  of  saltants  in  trans- 
ferring from  tube  to  tube.  All  of  the  numbers  listed  above  which  show 
constant,  though  but  slight,  differences  from  other  numbers  I  regard  as 
elementary  species,  the  Jordanons  of  Lotsy.  They  need  not  be  further 
characterized  or  differentiated  than  has  been  done  in  previous  pages.  One 
of  these  elementary  species,  H.  No.  3,  is  derived  from  an  Iowa  culture  prob- 
ably identical  in  character  with  that  from  which  Pammel,  King,  and  Bakke 
described  H.  sativum,  and  this  culture,  No.  3,  still  agrees  essentially  with 
their  description.  All  of  this  group  of  elementary  species  may  therefore 
be  regarded  as  belonging  to  the  Helminthosporium  sativum  group,  or  Lin- 
neon.  The  question  of  the  possible  identity  of  this  group  with  the  H.  teres 
and  H.  sorokinianum  groups  I  shall  not  now  discuss  further  than  to  point 
out  that  so  far  as  can  be  judged  from  the  picture  of  H.  sorokinianum  given 
by  Sorokin  (107)  that  species  is  not  characterized  by  longitudinally  eccen- 
tric conidia;  also  that  subsequent  to  the  publication  of  the  species  H.  sati- 
vum, Bakke  (6)  states  that  "cultural  experiments  have  determined  that  the 
disease  is  due  to  Helminthosporium  teres  Sacc."  He  adds:  "He  [Dr.  Ravn] 
further  substantiated  my  opinion  that  the  disease  was  due  to  H.  teres  and 
similar  to  what  had  been  so  prevalent  in  Denmark  during  the  years  1898 
and  1899."  Bakke,  in  a  foot-note,  however,  adds:  "A.  G.  Johnson,  of 
Madison,  Wisconsin,  considers  H.  sativum  and  H.  teres  distinct  forms." 
Saccardo,  who  examined  H.  sativum,  sent  to  him  by  Bakke,  expressed  the 


168 

opinion  that  the  disease  was  due  to  Helminthosporium  teres.  It  may  be 
remarked  here  that  Ravn  (91)  says  that  leaves  are  the  only  substrata  on 
which  conidia  are  developed ;  which  is  certainly  a  marked  distinction  from 
the  H.  sativum  group  which  sporulate  so  freely  on  agar  of  many  kinds. 

There  is  no  question  whatever  in  my  mind  that  by  meaira  of  biometry 
and  a  study  of  biologic  relations  and  cultural  characters,  tenable  distinctive 
diagnoses  can  be  drawn  up  for  many  races  of  Helminthosporium  on  the  five 
leading  cereals.  How  many  of  these  should  be  designated  by  binomials  and 
how  many  left  unnamed  appears,  on  final  analysis,  to  be  a  question  of  the 
utility  of  such  naming,  which,  in  turn,  may  hinge  upon  their  economic  or 
other  importance  rather  than  upon  the  magnitude  of  their  morphological 
or  other  differences. 

H.  No.  20  is  particularly  interesting  in  that  it  is — if  no  error  exists  in 
its  history — an  example  of  saltation  so  great  as  to  remove  the  organism 
entirely  from  the  group  under  discussion  (the  forms  with  tapering  conidia), 
and  consequently  to  place  it  in  a  group  (Linneon)  different  from  that  to 
which  its  known  relatives  (H.  Nos.  13  and  14)  belong. 

CONCLUSION 

The  present  study  was  undertaken  with  two  leading  objects:  (1)  to 
determine  the  efficient  cause  of  the  rotting  at  the  lower  part  of  the  wheat 
stem ;  and  (2)  to  throw  light  on  questions  of  morphology  and  parasitology 
in  the  genus  Helminthosporium.  The  questions  arising  from  saltation 
injected  an  additional  interesting  series  of  observations.  The  evidence  is 
complete  that  Helminthosporium  can  and  does  cause  foot-rot  at  the  base 
of  wheat  stems.  The  study  has  also  shown  the  Helminthosporium  (H. 
No.  1)  to  be  a  root  parasite.  This  phase  of  the  disease  has  been  studied 
only  incidentally,  but  it  is  worthy  of  searching  investigation  since  it  may 
lead  to  the  resetting  often  associated  with  foot-rot,  and  thus  predispose 
the  plant  to  foot-rot. 

SUMMARY 

1.  In  the  rotting  base  of  the  wheat  a  Helminthosporium  is  the  only 
organism  constantly  present  (p.  124). 

2.  The  culture  characters  of  this  fungus  were  studied  on  many  me- 
dia (p.  79)  and  under  many  and  various  environmental  conditions.     Slight 
changes  of  nutriment,  as  afforded  by  small  differences  in  agar  formulae  or 
by  the  temperature  at  which  the  agar  was  made,  produced  marked  effect 
on  growth-characters.    Of  many  agars  tried,  corn-meal  agar  proved    most 
useful.    Cereal  shoots  autoclaved,  served  as  a  still  more  favorable  medium. 


169 

3.  Morphological    characters   are   largely    altered    by   environment. 
Quantity' as  well  as  quality  of  food  produces  change  in  characters.     Humid- 
ity has  an   important   influence   on   the   production   of  conidia,   on   the 
aerial  mycelium,   and  on  sclerotial   formation  (p.    93),   influencing   even 
conidia  length  (p.  95). 

4.  The  optimum  temperature  for  growth  is  about  25°  (p.  98). 

5.  Carbohydrates  in  the  medium  favor  production  of  a  dark  color 
(p.  100). 

6.  Marked  effect  of  nutrition  conditions  on  conidial  length,  septation, 
and  shape  was  noted. 

7.  From  the  above  findings  it  follows  that  collections  to  be  compar- 
able must  be  made  under  similar  conditions  as  regards  the  factors  men- 
tioned (p.  102). 

8.  A  procedure  to  secure  standard  conditions  for  study  of  the  fungus 
was  devised  (p.  180). 

9.  The   mycelium,  aerial   and   submerged,    is   described.     The   cells 
bear  several   nuclei.      The  senescent  mycelium   undergoes    autodigestion 
(p.  108). 

10.  Conidia  show  distinct  basal  and  apical  markings.     The  wall  is  in 
two  layers:  the  outer  (episporium),  thin  and   brittle;  the   inner  (endospo- 
rium),  thick  and  gelatinous  (p.  111). 

11.  Germination  is  usually  terminal;  anastomosis  of  germ-tubes  is 
common  (p.  115). 

12.  The  conidia  are  thickest  at  a  point  between  the  base  of  the  conid- 
ium  and  its  middle  point.     The  concepts  '  'coefficient  of  longitudinal  ec- 
centricity" and  "coefficient  of  cylindricity"  are  introduced  for  purposes 
of  more  accurate  description  (pp.  117-120). 

13.  Conidial  length,  breadth,  and  septation  are  studied  biometrically. 

14.  For   comparison,   a   biometric  study  was   made  of   H.  ravenelii 
(p.  121). 

15.  The  etiological  relation  of  the  Helminthosporium  (H.  No.  1)  to 
foot-rot  was  demonstrated  by  its  constant  presence,  by  the  absence  of  other 
parasites,  and  by  its  proved  ability  to  cause  infection  and  rotting  under 
various  conditions,  as  by  inoculation  of  seedlings  in  Petri  dishes,  in  rag 
doll,  and  in  soil  (pp.  124-128).     The  fungus  was  shown  to  enter  cells  of  leaf- 
sheath,  stem,  and  root. 

16.  A  study  of  the  infection  phenomena  shows  important  changes  in 
the  cell-walls  of  the  host;  and  the  development  of  appresoria  and  a  callus- 
like  formation  (p.  128). 


170 

17.  Many  strains  of  Helminthosporium,  some  very  different  morpho- 
logically from  others,  can  produce  some  or  all  of  the  phenomena  of  infec- 
tion (p.  136). 

18.  Alternaria  produces  some  of  the  marks  of  infection,  including  the 
changes  in  the  host-cell,   the   "callus,"   and  entrance  info  the- host-cell. 
Penicillium  can  not  do  this  (p.  137). 

19.  Wheat,  corn,  barley,  rye,  sorghum,  Sudan-grass,  and  millet  are 
more  or  less  susceptible  to  rot  by  Helminthosporium. 

20.  Saltation,    possibly   mutation,    is   common   in   certain   races   of 
Helminthosporium  (p.  139). 

21 .  Saltation  is  evidenced  in  general  colony-character ;  rate  of  growth ; 
conidial  production;  conidial  clusters;  conidial  length,  breadth,  septation, 
and  shape;  mycelial  characters,  color,  zonation,  and  sclerotial  formation 
(pp.  141-144). 

22.  Certain  saltants  differed  so  markedly  from  their  parent  as  to 
far  exceed  the  usually  accepted  specific  limits  (p.  141). 

23.  Certain  correlations  and   tendencies  of  characters  in  saltation 
were  noted  (pp.  144-145). 

24.  The  saltants  were,  in  the  main,  permanent  in  character  (p.  145). 

25.  They  were  permanent  through  the  conidia  (p.  146). 

26.  What  appeared  to  be  reversions  sometimes  occurred  (p.  147). 

27.  Efforts  to  produce  saltation  artificially  failed  (pp.  147-148). 

28.  The  saltation  was  not  due  to  mixed  plantings,  and  can  not  be 
induced  by  implanting  or  wounding  (pp.  147,  148). 

29.  Saltations  are  not  due  to  parasites  (p.  148). 

30.  Saltations  in  abundance    were    derived    from    single-conidium 
cultures  (p.  149). 

31.  Saltation    is    very    frequent    as    compared    with    bud-variation 
noted  on  potatoes  and  tobacco  (pp.  150-151). 

32.  Numerous  variations  in  test-tube  cultures  are  reported  as  prob- 
able examples  of  saltations  (p.  152). 

33.  The  Helminthosporium  that  causes  wheat  foot-rot  belongs  to 
the  H.  sativum  group,  which  consists  of  a  large  number  of  elementary 
species  (p.  167). 


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APPENDIX 

METHODS 

For  measuring  conidia. — The  following  procedure  was  found  con- 
venient. An  ordinary  bacteriological  iridium  wire  was  plunged  into 
vaseline  and  then  so  laid  across  a  microscope  slide  as  to  leave  on  it  two 
complete,  narrow,  thin  streaks  of  vaseline  about  6  mm.  apart.  A  small 
drop  of  water  was  then  placed  between  these  two  vaseline  lines  and  the 
conidia-sample  added  and  evenly  distributed.  When  the  cover-glass 
was  placed  the  vaseline  prevented  the  conidia  from  scattering,  and  ren- 
dered it  possible  by  means  of  the  mechanical  stage  to  measure  every  conid- 
ium,  thus  securing  a  more  representative  sample  than  would  be  the 
case  if  some  conidia,  perhaps  of  some  particular  class,  were  allowed  to 
float  away. 

In  sampling  from  standard  cultures  for  purpose  of  conidia-measure- 
ment,  a  portion  of  a  shoot  about  6  mm.  long  that  was  evenly  and  densely 
covered  with  conidia  was  removed  to  the  slide.  Shoots  were  all  evenly 
and  abundantly  sporiferous  except  in  cases  where  entire  shoots  or  parts 
of  shoots  were  paler  and  bore  more  aerial  mycelium. 

To  avoid  unconscious  selection  in  measuring  conidia  a  mechanical  stage 
was  used,  and  all  conidia  encountered  in  certain  predetermined  posi- 
tions in  the  field  of  vision  were  measured.  Length  was  measured 
from  extreme  tip  to  extreme  base;  breadth,  at  the  thickest  point.  Meas- 
urements falling  exactly  between  two  classes  were  temporarily  so  recorded, 
and  later  distributed  equally  between  the  two  adjacent  classes. 

Measurements  for  coefficients  are  easily  made  by  projecting  the 
outline  of  the  conidium,  by  means  of  a  camera,  upon  quarter-section 
paper  of  convenient  ruling.  The  paper  may  readily  be  oriented  with 
the  conidium  in  any  desired  relation. 

The  rag  doll  for  inoculations. — An  adaptation  of  the  rag-doll  seed- tests 
was  found  useful  in  inoculations.  The  doll  was  made  of  a  strip  of 
cloth,  6X50  cm.  which  was  rolled  to  a  cylinder  about  6X2.5  cm.  and 
placed  in  a  test-tube  2.5X25  cm.  with  water,  and  autoclaved.  In  use 
the  roll  was  removed  to  a  sterile  Petri-dish  17  cm.  in  diameter,  the  water 
removed  to  the  desired  degree  by  wringing,  and  the  doll  unrolled  by  the 
use  of  sterile  forceps  (PI.  XXXIII).  Seedlings  raised  aseptically  were 


180 

then  laid  on  the  unrolled  doll  and  inoculation  made  in  the  desired  man- 
ner. The  doll  was  then  rolled  up,  inclosing  the  seedlings,  and  placed 
again  in  the  test-tube.  For  purposes  of  root  inoculation  the  doll  was 
suspended  in  the  test-tube  about  five  centimeters  from  its  bottom. 

Inoculations  in  soil. — In  addition  to  the  usual  pot  and  bench  inocu- 
lations, it  was  found  convenient  to  use  wide-mouth  vials,  12X70  mm. 
(see  page  128),  which  were  lined  with  stiff  paper  (so  cut  as  to  open  easily, 
PI.  XXXIII),  filled  with  soil,  and  autoclaved.  The  paper  envelope  with 
the  enclosed  soil  could  readily  be  withdrawn  from  the  vial,  and  opened  in 
order  to  insert  the  seed,  seedling,  or  inoculum,  and  later  repeated  exam- 
inations could  be  made  without  greatly  disturbing  the  plant. 

Imbedding  conidia. — Conidia  were  raised  under  standard  conditions 
(see  next  paragraph)  and  the  entire  shoot  bearing  them,  together  with 
the  adjacent  agar — a  strip  about  4  mm.  wide — was  removed  to  chrome- 
acetic  killing-fluid,  and  imbedded  in  the  usual  way. 

Procedure  to  secure  standard  conditions. — Petri  dishes  of  12  c.c.  washed 
agar,  when  solid,  were  inoculated  in  the  center  with  the  desired  organism. 
When,  in  the  course  of  a  few  days,  this  had  attained  a  colony-diameter 
of  2  to  3  cm.,  wheat  shoots,  autoclaved  in  water,  were  laid  on  the  surface 
of  the  agar,  the  basal  ends  of  the  shoots  touching  the  edge  of/the  advanc- 
ing colony.  Usually  about  six  shoots  were  used  per  plate,  resulting  in 
ample  material.  Aseptic  wheat  shoots  were  secured  by  the  method 
described  in  the  next  paragraph.  The  shoots  were  cut  for  autoclaving 
when  they  were  about  2-3  cm.  in  length.  This  medium  was  selected 
as  being  of  appropriate  composition  and  only  very  slightly  variable.  The 
washed  agar  in  uniform  quantity  in  Petri  dishes  of  the  same  depth  gave 
a  uniform  humidity,  while  the  mode  of  inoculation  was  also  uniform, 
doing  away  with  many  errors  that  arise  when  the  quantity  of  the  inoculum 
is  a  variable  factor. 

Growing  aseptic  seedlings. — Seeds  were  treated  three  hours  in  20% 
fresh  Javelle  water,  rinsed  with  sterile  distilled  water,  and  germinated 
on  damp  filter-paper  in  moist  chambers  (44).  In  the  latter  part  of  the 
study  para-toluene-sodium-sulphochloramide  was  substituted  for  Javelle 
water  in  seed-disinfection.  It  was  used  in  0.5%  aqueous  solution,  the 
seeds  being  immersed  for  twenty  minutes.  Such  preliminary  tests  as 
we  have  made,  indicate  that  a  solution  of  0.25  to  0.5%  is  efficient  as  a 
fungicide,  while  such  solutions  may  be  safely  used  without  injury  to  the 
grain.  It  certainly  possesses  value  for  such  uses  in  the  laboratory,  and 
may  be  of  service  as  a  fungicide  in  other  connections.  A  rather 


181 

extensive  test  of  its  utility  against  the  cereal  smuts  is  now  in  progress. 
Para  -  toluene  -  sodium  -  sulphochloramide  is  a  chemical  of  the  formula 
(p)CH3C6H4SO(NCl)ONa.  My  supply  was  secured  from  the  Abbott  Lab- 
oratories in  Chicago,  where  it  is  manufactured. 

Preparation  of  potato  plugs. — Instead  of  the  ordinary  potato  plug  it 
was  found  useful  to  place  a  glass  slip  in  a  test-tube  as  in  Fig.  2,  p.  94,  then  to 
crowd  into  the  tube  a  potato  slice,  bringing  it  into  contact  with  the  glass 
slip,  and,  at  its  angles,  with  the  walls  of  the  test-tube.  This  gave,  in 
addition  to  the  usual  surface  for  observation,  the  places  where  the  potato 
made  contact  with  the  glass  slip  and  with  the  walls  of  the  test-tube. 

A  device  for  the  study  of  humidity. — To  study  the  effect  of  changes  of 
humidity  on  conidiophores  and  conidia,  test-tubes  were  fitted  with  glass 
slips,  and  sterile  wheat  shoots  laid  across  them  (Fig.  2,  p. 94).  The  region 
1  is  of  about  90%  relative  humidity;  that  of  region  2  is  below  90%.  Com- 
bustion boats  full  of  agar  were  used  (Fig.  2)  to  secure  high  humidity 
for  the  whole  culture. 

LIST  OF  HELMINTHOSPORIUMS  USED  FOR 
PURPOSES  OF  COMPARISON 

H.  No.  1.  Isolated  by  F.  L.  Stevens  May  18,  1918,  from  wheat  dis- 
eased with  foot-rot,  from  Madison  Co.,  111. 

H.  No.  2.  H.  ravenelii,  isolated  by  F.  L.  Stevens  Jan.  17,  1920,  from 
specimen  received  from  A.  B.  Seymour,  collected  at  Lake  Charles,  La., 
Oct.,  1919,  by  E.  E.  Barnes.  This  specimen  was  thoroughly  typical, 
and  no  doubt  as  to  the  determination  can  be  entertained. 

H.  No.  3.,  labeled  H.  teres.  Received  from  A.  L.  Bakke  Jan.  5,  1920. 
Culture  isolated  Jan.  9,  1911. 

H.  No.  4.  Isolated  by  F.  L.  Stevens  Jan.  16,  1920,  from  specimens 
from  Iowa  labeled  H.  teres. 

H.  No.  5.  Isolated  by  F.  L.  Stevens  Jan.  16,  1920,  from  barley, 
from  specimen  from  Iowa  labeled  H.  gramineum. 

H.  No.  6.  From  E.  C.  Stakman,  Jan.  20,  1920.  Isolated  from 
blighted  seedling  of  Marquis  wheat. 

H.  No.  7.  From  E.  C.  Stakman,  Jan.  20,  1920.  Isolated  from 
Marquis  wheat. 

H.  No.  8.  From  E.  C.  S.  (E.  C.  Stakman),  Jan.  20,  1920.  Isolated 
from  Marquis  wheat  growing  in  sterile  soil. 

H.  No.  9.  From  E.  C.  S.,  Jan.  20,  1920.  Isolated  from  roots  of 
Marquis  wheat  seedlings. 


182 

H.  No.  10.  From  E.  C.  S.,  Jan.  20,  1920.  Isolated  from  barley, 
Madison,  Wis.,  "2-1919." 

H.  No.  11.  From  E.  C.  S.,  Jan.  20,  1920.  Isolated  in  1914  from 
wheat  in  Minnesota. 

H.  No.  12,  labeled  H.  gramineum,  from  E.  C.  S.,  Jan.  20, 1920.  Isolated 
from  barley,  Carver,  Minn.,  1914. 

H.  No.  13,  labeled  H.  sativum.  From  L.  W.  Durrell,  Feb.  6,  1920. 
From  barley  grown  on  the  agronomy  farm,  Ames,  Iowa.  Isolated  by 
Durrell  about  1918  "from  spots  or  lesions  pointed  out  as  typical  by  Dr. 'A. 
G.  Johnson." 

No.  14,  labeled  H.  sativum.  From  L.  W.  Durrell,  Feb.  6,  1920. 
From  barley.  Same  origin  as  No.  13. 

H.  No.  15,  labeled  H.  teres.  From  L.  W.  Durrell,  Feb.  6,  1920,  dated 
May  22,  1918.  Same  data  as  No.  13. 

H.  No.  16,  labeled  H.  teres.     Same  data  as  for  No.  13. 

H.  No.  17,  labeled  H.  gramineum.     Same  data  as  for  No.  13. 

H.  No.  18,  labeled  H.  avenae.  Same  data  as  for  No.  13,  except  that 
isolation  was  from  oats,  grown  in  rust  nursery. 

H.  No.  19,  labeled  H.  gramineum.  From  H.  Coons,  Feb.  16,  1920. 
Isolated  from  barley  in  1918. 

H.  No.  20,  labeled  H.  teres.  From  Centraal  Bureau  voor  Schimmel- 
cultures.  Culture  of  Feb.  11,  1920.  Isolated  from  late  blight  of  barley 
by  Bakke  and  sent  in  1914  to  Dr.  Westerdijk,  who  wrote  me  that 
"the  fungus  had  since  been  cultured  on  oatmeal  (roll  culture)  and  ears  of 
barley." 

H.  No.  21,  labeled  H.  inter seminatum.  From  Centraal  Bureau  voor 
Schimmelcultures,  March  12,  1920.  Culture  of  Feb.  11,  1920.  Received 
by  Dr.  Westerdijk  from  Miss  Dale  in  1912  and  cultured  on  oatmeal  or  on 
corn-meal. 

H.  No.  22.  From  Wanda  Weniger,  Mar.  24,  1920.  Isolated  April  28, 
1919,  from  kernels  of  Arnautka  wheat  in  North  Dakota.  Used  in  field 
studies  in  1919. 

H.  No.  23,  labeled  H.  teres.  From  Wanda  Weniger,  Mar.  24,  1920. 
Isolated  Feb.  27,  1920,  from  blade  of  barley  collected  at  Mandan,  N. 
Dak.  July  1919. 

H.  No.  24.  From  Wanda  Weniger,  Mar.  24,  1920.  Isolated  Feb. 
28,  1920,  from  first  node  of  Red  Durum  wheat  collected  at  Fargo,  N.  Dak., 
Aug.  1919. 


183 

H.  No.  25.  From  Wanda  Weniger,  Mar.  24,  1920.  Isolated  Nov.  20, 
1918,  from  blade  of  rye  collected  at  Fargo,  N.  Dak.  Used  in  field  studies 
in  1919. 

H.  No.  26,  labeled  H.  sativum.  From  Wanda  Weniger,  Mar.  24,  1920. 
Isolated  July  12,  1919,  from  blade  of  wheat  collected  at  Fargo. 

H.  No.  27.  From  Wanda  Weniger,  Mar.  24,  1920.  Isolated  from 
blade  of  "De"  wheat  collected  at  Fargo.  Isolated  Aug.  18,  1919,  and 
used  in  field  studies  in  1919. 

Cultures  22-27  were  grown  on  2  or  3%  potato-agar  with  2%  dex- 
trose from  the  time  of  their  isolation  until  received  by  me.  The  spe- 
cies determinations  were  stated  to  be  based  on  the  host;  not  on  morpho- 
logical characters. 

H.  No.  29.  From  G.  N.  Hoffer.  "Culture  B.  201a."  Isolated  from  a 
broken  corn-stalk  from  Ft.  Branch,  Ind.,  Aug.  13,  1919. 

H.  No.  30.  From  G.  N.  H.  (G.  N.  Hoffer).  "Culture  B.  180." 
Isolated  from  brown,  water-soaked  lesions  on  the  sheath  of  a  corn-leaf. 
I  lant  was  collected  at  Sullivan,  Ind.,  Aug.  12,  1919. 

H.  No.  31.  From  G.  N.  H.  "Culture  B.  170  A."  Isolated  from 
small  brown  spots  on  corn  leaves.  Collected  at  Delphi,  Ind.,  Aug.  8, 
1919. 

H.  No.  32.  From  G.  N.  H.  "Culture  B.  165."  Wras  isolated  from 
small  yellow  spots  on  corn  leaves  collected  at  Battle  Ground,  Ind.,  Aug. 
7,  1919. 

H.  No.  34.  From  G.  N.  H.  "Culture  B.  124."  Isolated  from  dark 
brown,  irregularly  shaped  lesions  on  corn  stalks  collected  at  Ames, 
Iowa,  July  25,  1919. 

H.  No.  36.  From  C.  E.  Kurtzweil.  Labeled  "3%  oat  206  5-40." 
Isolated  from  a  dead  corn-stalk  in  Tennessee. 

H.  Nos.  37  and  38.  Isolated  by  F.  L.  S.  (F.  L.  Stevens)  Aug.  8, 
1920,  from  wheat  grains  after  treatment  with  Javelle  water. 

H.  No.  39.  Isolated  by  F.  L.  S.  Nov.  20,  1920,  from  Chaetochloa 
(millet).  Conidium  short,  3-septate. 

H.  No.  40.  Isolated  by  F.  L.  S.  Nov.  20,  1920,  from  same  plant  as 
No.  39.  Conidium  long,  narrow. 

H.  No.  41.     Isolated  by  F.  L.  S.  Dec.  3,  1920,  from  sorghum. 

H.  No.  42.     Isolated  by  F.  L.  S.  Dec.  10,  1920,  from  millet. 

H.  No.  43.     Isolated  by  F.  L.  S.  Dec.  10,  1920,  from  sorghum. 

H.  No.  44.     Isolated   by  W.   L.    Blain   Dec.   20,   1920,   from  wheat. 

H.  No.  45.     Isolated  by  F.  L.  S.  from  association  with  H.  No.  44. 


184 

H.  No.  46.  From  E.  J.  Butler,  Mar.  10,  1921.  Isolated  from  wheat 
by  R.  E.  Massey  in  Khartoum  in  the  Anglo-Egyptian  Sudan.  The  state- 
ment is  made  that  "the  straw  was  completely  rotted  through  the  base,  and 
broke  off  short  when  handled.  The  fungus  was  present  in  pure  culture 
on  the  crown  and  roots."* 

DISCUSSION  OF  FOREGOING  LlST  WITH  SEVERAL 
BRIEF  DESCRIPTIONS 

H.  Nos.  29-32,  though  perhaps  not  quite  identical,  agreed  closely 
with  each  other.  They  were  mostly  5-celled,  with  the  central  cell  in- 
equilateral and  the  two  end-cells  pale.  In  conidial  measurements  they 
approached  rather  closely  to  H.  inaequalis  Shear,  H.  tritici  P.  Henn.,  and 
H.  geniculatum  T.  &  E. 

H.  Nos.  13  and  14  were  of  identical  parentage. 

H.  Nos.  15  and  16  were  from  one  strain  though  separated  by 
several  transfers.  The  original  strain  (15)  was  sent  because  the  growth 
(16)  did  not  look  characteristic. 

H.  No.  11  usually  gave  no  conidia  at  all  and  was  quite  distinct  in 
culture  characters  and  in  color  and  septation  of  conidia. 

H.  Nos.  36,  40,  and  41  are  closely  alike  in  morphological  characters. 
H.  Nos.  40  and  41  differ  from  H.  No.  36  in  that  they  do  not  possess  the 
abundant  aerial  mycelium.  Nos.  40  and  41  differ  in  zonation  and  in 
amount  of  aerial  mycelium,  and  all  three  of  these  forms  differ  somewhat 
in  their  conidial  graphs.  They  also  differ  in  mycelial  characters  and 
in  the  way  in  which  they  penetrate  wheat  cells,  though  all  three  do  the 
latter  vigorously,  completely  occupying  the  cells  and  causing  rotting. 
The  three  had  best  be  regarded  as  elementary  species  of  the  same  Linneon. 

Description  of  H.  No.  36. — Conidia  mostly  long  and  slender  (PI.  XXI, a) 
but  very  variable  in  length.  Stipe  short  but  distinct.  Apex  pale.  No 
constrictions  at  the  septa.  Septa  usually  thick  and  obvious.  Conidia 
tapering  very  slightly  from  point  of  maximum  thickness  toward  each  end; 
straight  or  slightly  arched.  Episporium  brittle;  endosporium  gelatinous. 
Conidiophores  uniform;  sterile  portion  thin,  slender,  quite  long  (350  ju), 
about  4  n  thick,  smooth,  brown,  cells  24-28  ju  long,  not  constricted 
at  the  septa;  fertile  portion  slightly  thicker  and  darker,  cells  short,  there- 
fore geniculations  crowded,  growth  on  agar  characterized  by  abundant 
aerial  mycelium  (PI.  X) — more  abundant  than  in  any  other  form  studied — • 
as  was  also  evident  under  standard  conditions. 


'From  letter  from  E.  J.  Butler  dated  Feb.  28,  1921. 


185 

Isolated   from   dead   corn-stalk   in   Tennessee   by   C.    E.    Kurtzweil. 
Data  on  conidial  length  of  H.  No.  36  are  as  follows: 

Frequency,  1  2         3         3         6        11       12       10       12       17       21        9 

Microns,  34  37.1    40.8   44.2   47.6     51  54.4   57.8    61.2    64.6     68     71.4 

Frequency,  10  15  12         11          7           6          3          3          0          2           1 

Microns,      74.6  78.2  81.8       85       88.4     91.8  95.2     98.6      102     105.4  108.8 

Conidial  breadth  ranged  uniformly  from  10.2  to  13.6  microns. 
Data  on  septa tion  are  as  follows: 

Frequency,      3  3  6          13         17          8          6  1  1 

Septa,  4          5  6  7  8          9          10         11         12 

Description  of  H.  No.  39. — Conidia  short,  quite  uniform  in  size, 
cylindrical  or  very  slightly  tapering  from  point  of  maximum  thickness 
(PJ.  XXI,  b).  Hilum  usually  evident.  Setpa  thick,  usually  three;  no 
constriction.  Apical  spot  barely  perceptible.  Episporium  brittle.  Endo- 
sporium  gelatinous.  Conidiophores  uniform;  sterile  portion  190-250  n  long, 
smooth,  3.5  ju.  thick,  not  constricted,  cells  about  17-24  ju  long;  fertile  por- 
tion much  darker  and  nearly  twice  as  thick  as  the  sterile  part,  genicula 
very  congested,  numerous,  usually  35-70  n  long.  Conidia  remaining  at- 
tached in  very  large  clusters. 

Data  on  conidial  length  are  as  follows: 

Frequency,   11         1         2        13       19       19       9         3         2         0         1 
Microns,       6.8   10.2    13.6     17     20.4    23.8    27.2    30.6     34     37.4   40.8   44.2 

Conidial  breadth  was  quite  uniformly  10.2  ju. 
Data  on  septation  are  as  follows: 

Frequency 3  1          31 

Septa 123 

This  organism  produced  on  wheat  many  infection  points  with  the 
appressoria  and  "callus,"  but  differed  from  H.  No.  1  in  the  minute  characters 
of  the  infection  spot. 

H.  No.  46  is  very  closely  like  H.  No.  39.  Data  on  the  conidial  length 
of  H.  No.  46  are  as  follows: 

Frequency 1  3          4          8          15          4  1 

Microns 8        20.4     23.8      27.2     30.6       34       37.4 

Conidial  breadth  was  uniformly  as  follows: 

Frequency 2          11          1 

Microns 6.8       10.2  13.6 

The  data  on  septation  are: 

Frequency 1  15 

Microns.  .  3 


GENERAL  EXPLANATION  OF  GRAPHS 

All  graphs  on  a  page  are  drawn  to  the  same  scale.  In  all  graphs  of 
length  and  breadth  the  class  value  is  3.4  /x.  All  computations  are  based  on 
class  values.  These  in  case  of  length  and  breadth  can  be  converted  to 
microns  by  use  of  the  factor  3.4,  or  by  the  following  table  of  equivalents: 

Class  Microns  Class  Microns  Class  Microns 

1  =  3.4  13  =  44.2  25  =     85. 

2  =  6.8  14  =  47.6  26  =     88.4 

3  =  10.2  15  =  51.  27  =     91.8 

4  =  13.6  16  =  54.4  28  =     95.2 

5  =  17.  17  =  57.8  29  =     98.6 

6  =  20.4  18  =  61.2  30  =  102. 

7  =  23.8  19  =  64.6  31  =  105.4 

8  =  27.2  20  =  68.  32  =  108.8 

9  =  30.6  21  =  71.4  33  =  112.2 

10  =     34.  22     =     74.8  34     =   115.6 

11  =     37.4  23     =     78.2 

12  =     40.8  24     =     81.6 

The  customary  symbols  are  used  in  presenting  the  data  of  the  graphs, 
f,  designating  frequency;  M,  mean;  o-,  standard  deviation;  and  CV,  co- 
efficient of  variability. 


—  P 
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P"  •  ft  g 

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_    P     CL 
WJ  !2    S' 


10    to    tO    to 

O    >-*    VO   \O 

H-    H-    If    H- 


O 


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tO    ts3 

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0^8 

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00    4^ 
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P     ^    3 

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. 

. 

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SO 
00 

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Oo    Oo    ^ 

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u      a 

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P   i   5j 

3    « 

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On    On 

O    00 


P     rt 

0.    3 


FIGURE  A 


GBAPH 


47.6 

51. 0 
54.4 
57.5 
61. £ 
64.6 

868.0 
o 

74.6 
76.  £ 

8  f.6 


66.4 

q  1.6 


96.6 


\ 


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7 


FIGURE  B 

Conidial  breadth  of  H.  No.  1  grown  on  corn-meal  agar 
made  at  different  temperatures:  Graph  5,  on  agar  made  at 
60°;  Graph  6,  on  agar  made  at  43°. 

Graph  M  a  .    CV 

5  5.50  ±  .04  .22  ±  .03  4.06  ±  .61 

6  5.50  *  .03  .18  ±  .02  3.31  =•=  .40 

Graph  6A,  conidial  breadth  of  H.  No.l  grown  under  standard 
conditions.  (See  app.,  p.  180). 

Graph        f  M  <r  CV 

6A        57          6.03  ±  .04  0.55  ±  .34        9. 13  ±.57 

Conidial  septa  of  H.  No.  1  grown  on  corn-meal  agar:  Graph 
7,  septa  on  agar  made  at  60°;  Graph  8,  septa  on  agar  made  at  43°. 

Graph  M  a-  CV 

7  7.30±.21  1.58  ±.14          21. 72  ±2. 12 

8  7.05  ±  .12  1.11  =fc  .09          15.86  =*=  1.31 


FIGURE  B 


-   ,-   ^          O 


H-    O 


•§• 


O  3 
P  2 


Iff 

S'  T3   orq 


*a-i>? 

fs : 


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O\    C/J    ON    H^ 

If    H-    tf    If 


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SB  ° 


to  »-»  to  •— 

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Cx>  to  to  to 

Cyi     *—     4-     to 

>-   O   ^J  ^* 


H-    H- 


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0\    p     NJ     H* 

i— *        ^^       H^        O^ 


P 


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3      ^ 


x.   o 

P*    £U        C^ 

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—       ^3    pj 

"S-o  'S  n 

_»  o   " 

10  n  ^ 
O  p*  CL 

3  5'  o 
•o  » 
5T  P  P 

5'^^ 
£ 


fr  fr  » 


O   Ov    *-»   ON 
OO    to    CA>    H-» 


^x   /^     P 

1M 

•      g     3_ 

p    P    n 
era   orq    O 


FIGURE  C 


GBAPH 


27  2 

30.6 
34.0 
37.4 
40.8 
44.2 
47.6 
51.0 
54.4 

57.8 
0 

61.2 
64.6 
68.0 
71.4 
74.8 
78.2 
81.6 
85.0 
88.4 
91.8 
95.2 


CD 


FIGURE  D 

Conidial  breadth  of  H.  No.  1  grown  on  green-wheat  agar  of  differ- 
ent   compositions:    Graph  13,  on    washed  agar  34,  green- wheat 
agar  %;  Graph  14,  on  washed  agar  %,  green-wheat  agar  y±. 
Graph         f  M  a  CV 

13  14  6. 10  ±.06  0.38  ±  .04  6.32  ±  .80 

14  44  5.98  ±  .07  0.71  ±  .05          11.87  ±  .86 

Conidial  septa  of  H.  No.  1:  Graph  15,  grown   on  washed  agar 
M,  green-wheat  agar  %;  Graph  16,  grown  on  washed  agar  %,  green- 
wheat  agar  }4. 
Graph         f  M  <r  CV 

15  65          3.83  ±  .18  2. 22  ±.13          58. 02  ±4. 10 

16  46         5.63  ±  .15  1.56  ±.11          27. 80  ±2. 10 


FIGURE  D 


Nat 


°O 

NO;  co  N 

—  cvj  OJ  CQ 

MICEON5 


Mai 


O— 


SEPTA 


FIGURE  E 


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FIGURE  F 


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3-     (T>     «      _  O 

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GRAPH  NUMBECS 
8 


37.2 
30.6 
34.0 
37.4 
40.8 
44.2 

51.0 
54.4 


71.4 
74.8 

78. 2  j 
SI. 6 
85.0 
88.4 
91.8 


102.0 

108.8 
112.2 


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Si 


FIGURE  G 


W 


GBAPH 


44.2 
476 
51.0 
54.4 
57.8 
61.  a 
84.6 
68.0 
71.4 

O  74.8 
JO 

:  78.  a 

i 

81.6 

85.0 

88.4 

91  8 

95.2 

98.6 

102.0 

105. 4- 

108.8 

I  18.2 


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FIGURE  H 


QBAPH 


20.4 

238 
27  2 
30.6 
34.0 
374 
40.8 
44.2 
47.6 
51.0 
54.4 

257. 

n 

061.2 
Z 

64.6 
68.0 
71  4 
74.8 
78.2 
81.6 
85.0 

884 
91  8 
95.2 
98.6 
102  0 


^       & 


ro  r\j 

N  OD 


\ 


\ 


\ 


FIGURE  I 


Length  of   1646  conidia  of  H.   No.    1   grown  on  corn-meal  agar. 
See  also  pp.  120-121. 

Graph                          M  a                                  CV 

34                    14.34  =t  .08  5.35  =*=  .06                 37.35  =*=  .70 


FIGURE  J 


N0.35 


VO   b-  oo  O)   O 

v5EPTA- 


Conidial  septation  of  H.  No.  1. 

Graph  f  M  <r  CV 

35  58  7.91  ±  .08  .99  ±  .—  12.51  *  .79 


FIGURE  K 

Graphs  of  conidial  length  of  H.  No.  1  grown  under  standard  con- 
ditions (see  app.  p.  180):     Graphs  36-40  represent  respectively  plates 
a-e;  Graph  41  represents  plate  e';  and  Graph  42  is  a  composite  of  plates 
a,  b,  c,  d,  e'  (see  p.  120). 
Graph        f  M  a  CV 

36  123  23.21  ±  .15  2.36  ±  .10  10.02  ±   .46 

37  107  22.51  ±  .15  2. 54  ±.10  11. 28=*=. 49 

38  142     22.59  ±  .16      2.87  ±  .11     12.70  ±  .51 

39  180     22. 42  ±.17      3. 42  ±.12     15. 25  ±.55 

40  21.18  ±  .18      3.55  ±  .13     16.80  ±  .63 

41  647     22.71  ±  .05      2.26  ±  .04      9.95  ±  .01 

42  1199     22.62  ±  .05      2. 76  ±.03     12.22±.1£ 


FIGURE  K 


— <r  <    —  in  co  ~  «r   co 


FIGURE  L 

Graphs  of  conidial  length  of  H.  No.  2  (H.  ravenelii). 

Graph  43,  Seymour  and  Earle,  Economic  Fungi,  No.  399,  Florida, 
1890. 

Graph  44,  Ellis,  North  American!  Fungi,  No.  368.     North  Carolina. 

Graph  45,  A.  B.  Seymour's  specimen  as  grown  by  me  on  corn-meal 
agar. 

Graph  46,  deThu  men,    Mycotheca    Universalia,    No.    1468.    Caro- 
lina, 1876. 

Graph  47,  A.  B.  Seymour's  specimen  from  Louisiana,  1919. 

Graph  48,  Bartholomew,    Fungi    Columbiana,    No.    3026.       Nova 
Scotia,  1909. 

Graph  49,  Ravenel,  Fungi  Americani  Exsiccati,  No.  165.     Florida. 

Graph  50,  Ellis  and  Everhart,  Fungi  Columbiani,  No.  4633.     Flori- 
da, 1914. 

Graph  51,  Ellis  and  Everhart,  Fungi  Columbiani,  No.  465.     Caro- 
lina, 1894. 

Graph  52,  Rabenhorst,    Fungi    Europeai,    No.    3082.      Argentine, 
1878,  sample  1. 

Graph  53,  Rabenhorst,   Fungi   Europeai,    No.   3082a.      Argentine, 

1878,  sample  2. 
Graph  M  a  CV 

43  14.97  ±  .18  2.53  ±  .13  16.89  ±     .89 

44  14.80  ±  .22  3. 58  ±.15  24. 17=*=  1.73 

45  14.79  ±  .14  2.27  ±  .10  15.34  ±     .69 

46  14.49  ±  .19  2.19  ±  .16  15.11  ±     .97 

47  14. 38  ±.  16  2. 38  ±.11  16.54  ±     .84 

48  14.35  ±  .22  3. 02  ±.15  21. 04  ±1.15 

49  14.34  ±  .21  2. 74  ±.15  19. 10  ±1.18 

50  13.98  ±  .20  2.82  ±  .14  20.15  ±  1.50 

51  13.78  ±  .20  3. 49  ±.14  25. 31  ±1.08 

52  13.02  ±  .20  3. 16  ±.14  24. 25  ±1.18 

53  12.05  ±  .19  3.10  ±  .13  25.  72  ±    1.22 


FIGURE  L 


43 


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48 
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47.6 
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FIGURE  T 


FIGURE  U 

Graphs  of  conidial  length  of  several  Helminthosporiums  under 
standard  conditions:  Graph  93,  H.  No.  14;  Graph  94,  H.  No.  13; 
Graph  95,  H.  No.  15;  Graph  96,  H.  No.  16;  Graph  97,  H.  No.  17;  Graph 
98,  H.  No.  18;  Graph  99,  H.  No.  19;  Graph  100,  H.  No.  20. 

Graph       f  M  a  CV 

93  404     22.04  =t  .10      3.03  ±  .07     13.76  =•=  .33 

94  597     24.78  =t  .09      3.53  ±  .06     14.24  ±  .28 


95 

461 

22 

54  ± 

.10 

3 

.49  ± 

.07 

15 

.51  ± 

.35 

96 

445 

23 

75  =*= 

.12 

3 

.80  =t 

.08 

16 

.03  ± 

.37 

97 

252 

24 

39  ± 

.15 

3 

.63  ± 

.10 

14 

.88  ± 

.45 

98 

97 

23. 

03  =b 

.28 

4 

.10  =*= 

.19 

17 

.81  ± 

.88 

99 

205 

24. 

59  ± 

.19 

4 

.15  ± 

.13 

16 

.89  ± 

.57 

100 

315 

18. 

84  =t 

.12 

3 

.30  ± 

.08 

17 

.53  ± 

.48 

FIGURE  U 


FIGURE  V 

Graphs  of  conidial  breadth  of  several  Helminthosporiums  under 
standard  conditions:  Graph  101,  H.  No.  11;  Graph  102,  H.  No.  20; 
Graph  103,  H.  No.  13;  Graph  104,  H.  No.  14;  Graph  105,  H.  No.  15; 
Graph  106,  H.  No.  16. 


Graph 

f 

M 

o-                           CV 

101 

39 

5.19 

d=  .04 

.40  ± 

.03 

7.74  ^ 

=     .59 

102 

58 

5.11 

±  .14 

.52  ± 

.03 

10.27  =i 

=     .65 

103 

79 

5.97 

d=  .08 

1.06  ± 

.05 

17.85  d 

=     .98 

104 

32 

5.59 

d=   .08 

.74  ± 

.06 

13.30  =" 

=  1.14 

105 

88 

5.39 

d=   .04 

.56  d= 

.02 

10.50  d 

=     .53 

106 

45 

5.88 

±  .05 

.56  ± 

.04 

9.61  d 

=     .68 

FIGURE  V 


?  » 

SSSSS   =0 

sf    o  n 


§OJ    — 
O    H- 


i-1    K)    KJ    K3 

O    o   —   ^* 


H-    |f    H-    H-   S    "  TO 

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H-    H-    H-  < 


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?1 

Si 

s| 
a 

? 


FIGURE  W 


QBAPH  NUMBEES 
I 


FIGURE  X 

Graphs  of  conidial  septation — under  standard  conditions — of   H. 
No.  la  (111);  H.  No.  Ib  (112);  H.  No.  Ic  (113). 


Graph 

f 

M 

Or 

CV 

111 

49 

6 

.71  ± 

.10 

1 

.12  ± 

.07 

16. 

75 

=t  1 

.17 

112 

65 

6 

.61  =*= 

.09 

1 

.15  ± 

.06 

17. 

52 

±  1 

.08 

113 

17 

6 

64  ± 

.09 

,58  =*= 

.06 

8. 

85 

±  1 

.02 

FIGURE  X 


FIGURE  Y 

Saltant 

Graph   f 

M 

a 

CV 

Ml-5 

114 

160 

23, 

40  =«= 

.18 

3. 

53  =±= 

.13 

15. 

11  ± 

.58 

M2-4 

115 

154 

21. 

77  ± 

.17 

3. 

17  ± 

.12 

14. 

56  =*= 

.57 

M3-4 

116 

131 

22. 

66  * 

.20 

3. 

50  =•= 

.14 

15. 

48  ± 

.66 

M4-6 

117 

168 

22. 

,72  ± 

.21 

4. 

04  ± 

.14 

17. 

80  =*= 

.67 

M5-5 

118 

145 

23. 

72  ± 

.19 

3. 

52  ± 

.13 

14. 

86  ± 

.60 

M6-5 

119 

166 

23. 

65  ± 

.20 

3. 

85  * 

.14 

16. 

28  ± 

.61 

M8-8 

120 

156 

23. 

92  ± 

.18 

3. 

38  ± 

.12 

14. 

35  ± 

.55 

M12-3 

121 

148 

23 

64  =t 

.20 

3. 

73  ± 

.14 

15. 

81  =t 

.63 

M13-3 

122 

173 

22 

.73  ± 

.18 

3. 

61  ± 

.13 

15. 

90  ± 

.69 

M14-3 

123 

126 

22 

,32  ± 

.17 

2. 

98  ± 

.12 

13. 

37  ± 

.57 

M15-3 

124 

134 

23 

,51  ± 

.20 

3. 

51  =*= 

.14 

14. 

93  ± 

.62 

M36-2 

125 

156 

22 

.31  =b 

.16 

3. 

14  =t 

.12 

14. 

09  ± 

.54 

M37-2 

126 

131 

22 

03  ± 

.21 

3. 

64  ± 

.15 

16. 

55  ± 

.70 

M32-2 

127 

86 

23 

00  ± 

.16 

2. 

33  ± 

.12 

10. 

14  ± 

.52 

M33-2 

128 

155 

22 

,37  ± 

.16 

3 

02  ± 

.11 

13. 

52  ± 

.52 

M40-2 

129 

97 

17 

.70  =*= 

.17 

2 

51  ± 

.12 

14. 

19  ± 

.70 

M41-2 

130 

148 

22 

.92  ± 

.19 

3 

.45  ± 

.13 

15. 

05  ± 

.60 

M42-2 

131 

125 

22 

.36  ± 

.21 

3 

.53  ± 

.15 

15. 

81  ± 

.69 

M43-2 

132 

139 

22 

.00  ± 

.19 

3 

.36  ± 

.13 

15. 

29  =t 

.63 

M44-2 

133 

134 

22 

.18  ± 

.15 

2 

.72  ± 

.11 

12. 

27  ± 

.51 

M45-2 

134 

142 

22 

.88  =*= 

.21 

3 

.76  ± 

.15 

16. 

43  ± 

.67 

M46-2 

135 

158 

22 

.84  ± 

.17 

3 

.34  ± 

.12 

14. 

66  =»= 

.56 

M47-2 

136 

112 

23 

.53  ± 

.22 

3 

.56  ± 

.16 

15. 

12  ± 

.69 

M48-2 

137 

146 

22 

.00  ± 

.16 

3 

.02  ± 

.11 

13. 

78  ± 

.55 

Ml  7-3 

138 

128 

22 

.56  ± 

.14 

2 

.38  =*= 

.10 

10. 

56  ± 

.45 

FIGURE  Y 


114 
fl5 
116 
117 
116 
1 19 
120 

121 

\Z 
123 

124 


S  l25 

I l26 

I . 


130 
131 

13£ 
133 


134 
135 
«36 


1381 


X 


7 


\ 


X 


\ 


\ 


\ 


\ 


N 


\ 


\ 


V 


x 


\ 


\ 


X 


—  ^-oo— •  in  GO  — 


t  co  w 

^  (O  CU 


PLATE  VII 


Several  wheat  stems  showing  characteristic  diseased  spots; 
also  diseased  portion  at  the  node  in  one  shoot. 


PLATE  VIII 


Diseased  plant,  showing  numerous  dead  leaves  and 

leaf-sheaths,  also  more  than  a  dozen  new  shoots 

issuing  from  below  the  diseased  portions. 

These  shoots  varied  in  height  from  a  few 

millimeters  to  several  centimeters. 


PLATE  IX 


^22 


H.  Nos.  1,  3,  4,  5,  20,  and  22,  growing  on  corn-meal  agar. 


PLATE  X 


H.  No.  36,  showing  very  floccose  mycelium. 


PLATE  XI 
H.  No.  3  (left)  and  No.  1  (right)  as  grown  in  Piorkowski-flask  culture. 


PLATE  XI 


PLATE  XII 
H.  No.  1  as  grown  in  Kolle-flask  culture. 


PLATE  XII 


PLATE  XIII 
H.  No.  3  as  grown  in  Kolle-flask  culture. 


PLATE  XIII 


PLATE  XIV 

Petri-dish   cultures  of   H.    No.    1    on   different   amounts   of  agar:    14,   on    12   c.c. ; 
15,  on  30  c.c. 


PLATE  XIV 


14 


15 


PLATE  XV 


H.  No.  1  growing  in  tubes  of  rice  with  different  amounts  of  water. 
Note  abundance  of  sclerotia  in  the  drier  tubes  at  the  left. 


PLATE  XVI 

Tj 

Braz.il- nuts 


TflUDl 


H.  No.  1  grown  on  washed  agar  with  nutrients  added  as  indicated— 
fragments  of  Brazil-nuts,  rice,  tapioca,  and  corn-meal  agavy    (Circles 
indicate  approximate  limits  of  growth  at  ' 


PLATE  XVII 


Photomicrographs  of  H.  No.  1,  showing  attachment 
of  conidia  to  conidophores. 


PLATE  XVIII 


Photomicrographs  of  H.  No.  1,  showing  the  fragile  nature  of  the  outer  brown"] 
spore-wall  and  the  gelatinous  texture  of  the  hyaline  mass  enclosed. 
(Three  different   magnifications.) 


PLATE  XIX 


;;v>  Y':«*  '**-»«> 

J5*/    \  &* •  * 

a   */  '        *K^ 

-  '  ',  -  *  v  * 
*  \  *? 


f 


•«3MH'g:       ^-  -' 


* 


Photomicrographs  of  H.  No.  1,  showing  conidia  under  different  magnifications. 


PLATE  XX 


Photomicrograph  of  conidia  of  fl".  ravenelii. 


PLATE  XXI 


Conidia  (a]  of  H.  No.  36,  showing  variation  in  size  and  shape; 
b  and  c,  conidia  and  a  conidiophore  of  H.  No.  39. 


PLATE  XXII 


Two  saltants:  upper  one  showing  origin  of  M5;  lower  one 
showing  a  white  clump  and  slow  growth. 


PLATE  XXIII 


Two  saltants:  upper  one  showing  origin  of  Ml;  lower  one 
of  slow  growth  and  bearing  clumps. 


PLATE  XXIV 


Upper  figure  showing  origin  of  M2;  lower  figure 
showing  origin  of  M30-M34. 


PLATE  XXV 
Saltants  growing  with  their  respective  originals. 


PLATE  XXV 


PLATE  XXVI 

Photomicrographs  (same  scale)  of  conidia  of  several  Helminthosporiums: 
a,  H.  No.  1;  6,»M6;  c,  M35;  d,  H.  20. 


PLATE  XXVI 


PLATE  XXVII 


Saltants  growing  with  their  respective  originals. 


PLATE  XXVIII 


M34,  characterized  by  abundance  of  sclerotia  and  white  mycelial  clumps, 
the  latter  a  constant  character  of  this  saltant. 


PLATE  XXIX 

Above,  H.  No.  1  wounded  by  hot  wire  at  points  shown; 
below,  H.  No.  1,  with  H.  No.  1  implanted  at  various  distances 
within  and  without  the  colony. 


PLATE  XXIX 


PLATE  XXX 

Above,  two  implants  of  H.  No.  1 — one  of  them  the  origin 
of  M70 — in  H.  No.  1  colony,  showing  some  white  floccose  aerial 
mycelium;  below,  M26,  with  origins  of  M53,  M56,  and  M57. 


PLATE  XXX 


PLATE  XXXI 
\126-1  as  it  appeared  on  two  separate  plates. 


PLATE  XXXI 


n  26-r 


PLATE  XXXII 

M125.    Pale  colonies,  showing  dark  sectors  which  were  apparently  reversions 
to  the  original  form. 


PLATE  XXXII 


PLATE  XXXIII 

Showing  method  of  using  rag  doll  in  inoculations  (a,  b,  c)  and  also  (d)  vial  and 
paper  cylinder  for  soil  inoculation:  a,  the  rag  doll  unrolled  in  sterile  Petri-dish,  and 
aseptic  wheat  seedlings  in  place,  ready  for  inoculation;  b,  doll  in  place  in  tube 
and  seedlings  growing;  c,  showing  development  of  root  hairs  in  condition  for  inocu- 
lation below  the  doll;  d,  as  stated  above. 


PLATE  XXXIII 


PLATE  XXXIV 


Rag  doll  opened  for  examination  6  days  after  inoculation.     All  the  seedlings 
show  beginnings  of  foot-rot. 


U.C.BERKELEY  LIBRARIES 


881261 


THE  UNIVERSITY  OF  CALIFORNIA  LIBRARY 


